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User Manual DeCyder Differential Analysis Software, Version 5.0

1. 18 8 8 8 5 2 S 8 2 S Gl lll Upon completion of the batch run the DIA XML BVA and pick list files are automatically generated in the requested folders The preparative gel and pick list can then be taken to the appropriate Ettan spot picking instrument for automated spot excision However it is recommended that the BVA workspace is reviewed prior to generating the pick list A workflow of using the Batch Processor to assign proteins of interest only is therefore recommended This involves following the above tutorial but only selecting the Protein of Interest check box without selecting Pick status in the Protein Filter dialog box DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 235 Tutorial IV Fully automated identification of differentially expressed proteins A then avoiding generating a pick list by cancelling the Set Pick List Filename dialog box In this way the subsequent BVA file will have protein differences highlighted as proteins of interest which can be manually reviewed then confirmed before assigning the proteins of interest with a Pick status see chapter 12 q A 236 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Appendix A Recommended workflow for analysis of multiple gels The following workflows describe the various recommended st
2. 5 Master No Status Protein 1D Protein AC Ttest 3 9e 010 20 20 A MIP P SpotMap Protein ID Protein AC Name Comment sy fy I I j m Ready I Pick M PTM Confirm Da I Protein of Interest Data Control Panel Focus Secondary 20 Vew NM There is also a data control panel present on the bottom of the workspace which incorporates specific functionality features The contents of the control panel and the Table View are dependent on the mode selected 4 1 2 Structure The BVA is composed of four different modes which display data manipulated through various tables and associated controls Each mode provides different functionality associated with specific processes in BVA analysis Spot Map Table The Spot Map table is used to set up images for spot matching and statistical analysis The table lists data related to the Spot Maps imported from DeCyder Differential Analysis Software DIA module Match Table The Match table is used for the processes associated with inter gel matching The table lists all data associated with the matching algorithm DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN Protein Table PT The Protein table is used to display and process data associated with the protein spots identified across the gels Each row of the table corresponds to one protein s
3. Places My Network File name Gel 01 Cy2 Standard gel Files of type Gel image files tif gel X Cancel 5 Select Gel 01 Cy3 Control then click Open in the Load Secondary Gel Image window 6 Select Gel 01 Cy5 Treated then click Open in the Load Tertiary Gel Image window 11 4 2 Spot detection and quantitation 1 After the images have been loaded select Process Process Gel Images He Process Gel Images Exclude Filter Re Normalise Protein Filter Unassign all Protein of Interest Assign all Protein of Interest as Pick 2 Enter the value 2500 for the estimated no of spots see section 3 3 1 for an explanation for this value Then click OK to begin spot detection and quantitation on all three images 176 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial Il Employing an internal standard to Analyze Protein Changes Oy Process Gel Images F Algorithm Selection Version 5 00 01 Description Co detection algorithm Performs spot detection in one or more images based on features in the image contrast Estimated no of spots 2500 Autodetect Picking references I Cancel Help The DIA analysis takes between 3 and 10 minutes depending on the specifications of the computer 11 4 3 Viewing the DIA workspace 1 Upon completion of the DIA processing the spot map data is displayed in the workspace as shown below NM Decyder DIA Control Treated dia File Edt
4. oococccccccccccccoonnccnonononn 251 Appendix D Experimental design and set up examples 257 Appendix E DeCyder Differential Analysis Software keyboard shortcuts 267 Appendix F Related products and documentation 271 Appendix G Glossa Vi iii iba 275 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Introduction to DeCyder Differential Analysis Software er 1 Introduction to DeCyder Differential Analysis Software 1 1 Introduction Two dimensional electrophoresis 2 D electrophoresis is a leading tool in proteomics research today capable of visualizing many components of complex proteomes in a single gel Ettan DIGE Difference Gel Electrophoresis system is a method for pre labelling protein samples prior to 2 D electrophoresis The system is based upon the specific properties of CyDye DIGE Fluor dyes which enable multiplexing of separate protein mixtures on the same 2 D gel DeCyder Differential Analysis Software is an automated image analysis software suite which enables detection quantitation matching and analysis of Ettan DIGE system gels The software was developed as part of Ettan DIGE system to exploit the multiplexing capabilities of the CyDye DIGE Fluor dyes Multiplexing the co migration of more than one sample per gel enables the inclusion of an internal standard The internal standard is used to derive statistical data within and between gels This experimental design us
5. ccccccccnccccininoninoninnnns 107 5 3 Identification of reference markers cccccececeeceeeeeeeeeeeeeeeee 108 5 4 Identifying Proteins of Interest ccccnncccccccnonooccnnnnnononannnnonnnos 109 5 4 1 Identifying proteins for picking using the DIA module 110 5 4 2 Identifying proteins of interest using the BVA module 112 5 5 Assigning Spots for PICKING secta in 114 5 6 Editing Pick HKOCAONS a Sid 116 5 7 Generating a Rick Lia Dia 119 6 Batch processor O 121 6 2 Batchillstcraai ia do Na 121 6 2 1 Setting up the DIA Batch List ooonooccoccncconanacconononananonononanos 122 6 2 2 Setting up the BVA Batch List ooooococcccconocoaoncconoononononononanos 125 6 3 Editing the batchi st reann tnn rada 126 6 3 1 DIA patchit anene iaa ido dd 126 632 BVA Dalla ii i 126 64 Saving Data teta tato tinta 127 6 5 Running the batch processor cocccoccoooooocccnnnnnnnncnnnnnnnnnnnnaninaninnnos 128 7 XML Toolbox A 2 abe e Tins Bes en nn a icles einen tes eonecaties 129 7 2 Opening the XML Toolbox module ooooooocccccccncoccnccccioooonnnncononos 129 7 3 EXCUSA AA ee 130 TA A ethane sPAL ALS A 132 7 4 1 BVA parameters reiia a ra rene a skt 132 7 4 2 DIA parametee FS aa att ia Ian 135 8 LWS Integration O 139 8 2 Ettan LWS with samples generated within Ettan DIGE system 140 8 3 Enter a protein tube generated within Ettan DIGE system NOEN WS a o aa ce 142 8 4 Enter a gel prepared for spot picki
6. XML Toolbox Comprises tools for the extraction of data from the different XML files produced in DeCyder Differential Analysis Software DIA DeCyder DIA processes a set of gel images each saved as either 16 bit TIFF or customized GEL files from a single gel Each image in the pair is generated from samples labelled with different fluors Images must be processed in the DIA interface prior to data analysis in BVA The DIA algorithms detect spots on a combined image derived from merging individual images from an in gel set of images This co detection ensures that all spots are represented in all images DIA then quantitates spot protein abundance for each image and expresses these values as a ratio thereby indicating changes in expression levels by direct comparison of corresponding spots The data can be saved as a DIA file from which spot pick lists can be exported as a text file In addition data can be exported in an XML format which can be queried using the XML toolbox for multi gel analysis in DeCyder BVA or copied directly from DIA and pasted into applications such as Microsoft Word and Excel DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 155 ED Tutorials Introduction 2 D DIGE gel Gel 1 Gel 2 Gel 3 Gel 4 156 Pick list for a single gel experiment TXT Data extraction using XML Toolbox gt a A o Nx fl Multi gel analyses using BVA DIA file Fig 9 1 Schematic rep
7. Detected 2340 ca Histogram View 2 Number of Spots o A 3 GIB Wad XOW Excluded Picked T Peak Height Ratio Pick pos Aiea KIGWE as pesnisosa 542 Fick pos Status spot No Unconfirmed 571 Unconfirmed 570 Unconfirmed 569 Unconfirmed 568 Unconfirmed 567 Unconfirmed 566 Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Hneanfirmad Abundance Similar Similar Similar Similar Similar Similar Similar Similar Similar Similar Similar Similar Similar Excluded Volume Ratio Spot No Position Table View E Protein ID F Pick I PIM T Protein of Interest J Exclude Confirm Focus Primary 3D View NUM The four views are all linked Clicking on a spot on Image View highlights the spot in magenta in the Histogram View the Histogram View is disabled when analyzing single images such as SYPRO stained gel images The spot is also represented three dimensionally and is highlighted in gray in the Table View Q 3 To display the entire gel image click on the Fit to window icon The detected spots are of three types increased decreased and unchanged in expression colored blue red and green respectively in the Histogram View If after detection the hourglass turns back to a cursor the Table View remains empty click on the Properties ic
8. ED BVA Biological Variation Analysis Module 82 from independent factors such as time temperature and dose These are entered as conditions 1 and 2 The ANOVA tests require a minimum of two replicate data points Greater statistical validity can be achieved using larger number of replicates It is recommended that the largest possible number of biological replicates are performed for optimal validity However if biological replicates are not possible gel replicates can be used for the purposes of the statistical analysis In these instances it is recommended that gels be run at least in triplicate hence each group has at least three data points As with the Student s T test the ANOVA tests can be either independent or paired Paired ANOVA tests require dependencies to be applied see section 4 8 4 Null hypothesis The ANOVA test null hypothesis is that there is no change in the protein abundance between any of the experimental groups analyzed The ANOVA test compares the variance between groups with the variance within groups The ratio of the between groups variance to the within groups variance is known as the F ratio and can be used to generate a p value If the F ratio is large then it shows that the variation between groups is large compared with the variation within groups and thus that the groups may be different Therefore the p value displayed in the ANOVA column of the protein table is a measure of the probabilit
9. Include in Match Table IV Unmatched IV Automatic Match Level 1 IV Automatic Match Level 2 IV Manually Matched Spots IV Added Cancel Include in Match Table Selects the categories of spots that are to be displayed in the Match Table 70 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN 4 7 Graphical representation The graphical view in DeCyder Differential Analysis Software BVA allows the user to view scatter plot representations of data points associated with individual proteins For example the manner in which the expression of a protein changes with time or drug dosage can be graphically viewed The graphical views can only be seen in the Protein Table mode and Appearance Table mode Examples of the graphical representations are illustrated in the next section Properties associated with graph view are defined in the Graph View dialog box Selecting View Properties or selecting the Properties icon then selecting the Graph View tab displays the Graph View Properties dialog box DeCyder BYA Properties Spot Map Table Match Table Protein Table Appearance Table Image View 3D View Graph View Database Colors Parameter Visualization X axis Group zl Y axis Standardised Log Abundance Dashed Line Gel No y Spot Colour Group Include in Graph View IV Spots IV Mean Value Crosses IV Lines
10. Type No of Spots Matched Function Group Group Description Condition Condition2 Sample ID Pending Gel 04 Cy2 Standard gl DIGE Min 2298 Standard Pending Gel 04 Cy3 Treated gell DIGE Min 2298 Unassigned Pending Gel04 Cy5 Control gel 1 DIGE Min 2298 Unassigned Pending Gel 01 Cy2 Standard g2 DIGE Min 2254 Standard Pending Gel 01 Cy3 Control gel 2 DIGE Min 2254 Unassigned Pending Gel01 Cy5 Treated gel DIGE Min 2254 Unassigned Master Gel 02 Cy2 Standard g DIGE Min 2572 Standard Matched Gel 02 Cy3 Treated gel DIGE Min 2572 Unassigned Matched Gel 02 Cy5 Control gel3 DIGE Min 2572 Unassigned Pending Gel 03 Cy2 Standard g4 DIGE Min 2507 Standard PREZ E PEED Function for Spot Map Gel 03 Cy5 Treated gel Group Sample ID Spot Map Comment Analysis 4 I Master M Template T I Pick P Unassigned y Focus Spot Map Table 2 Under the column Function all the images will be labelled with the function Analysis A and one of the analysis images the image with the largest number of detected spots will be labelled with the function Master M The analysis function designation indicates that each of the images will be included in post matching statistical analysis The Master function can be assigned to a different image by selecting the image to be assigned as master using the left mouse click Tick the box entitled Master at the bottom of the table in the area entitled Function for Spot Map 11 5 3 Expe
11. 6 If the spot on the match image has also been wrongly matched break this match as before Select the corresponding spots on the master image and the match image and click on the button entitled Add Match 7 This procedure can be carried out for example using 5 randomly chosen Level 1 spots and 10 level 2 spots these spots should be real protein spots If the matches are accurate in each case move to the Protein Table PT If the majority of these 15 matches were incorrect it may be necessary to perform landmarking and a further round of matching The purpose of the above step is to get an overall impression of the matching accuracy This can be done in several ways One way is to look at the match vectors for each gel if the match vectors are not oriented in the same direction over the gel this may indicate an area of mismatches For example if in one area of a gel the match vectors are at right angles to the other match vectors on the gel then this area should be landmarked and re matched Rather than choosing a random auto level 1 match and flicking through each gel for that protein on a gel by gel basis click on random spots on the image spread around the gel and see if they are correctly matched If the majority are matched correctly then you can move on to the next gel DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial Il Employing an internal standard to Analyze Protein Changes Oy FE DeCyder
12. BVA Ecoli Con treated bva File Edit View Process Help B rs errar So z 4 e 18 0 8 A aaa Ne Match Table Type Unconfirmed 817 694 Auto Levelt Unconfirmed 806 1183 Auto Level2 Unconfirmed Unmatched Unconfirmed 810 827 AutoLevell Unconfirmed 810 1021 Auto Level2 Unconfirmed 808 1159 Auto Level2 Unconfirmed Unmatched Unconfirmed uto Level1 Unconfirmed 814 851 Auto Level2 Unconfirmed Unmatched Unconfirmed 811 1228 Auto Level2 Unconfirmed Unmatched Unconfirmed Unmatched Unconfirmed 817 602 Auto Level2 Unconfirmed Unmatched Spot No 1775 Position 809 763 Spot No 1733 Position 821 732 Match Image Master Spot Landmark mode Unmatched Spot Master Spot Match Comment jj Confirm Match Break Match i Remove Focus Master Image 11 7 Post matching landmarking If rematching is necessary a post matching landmark procedure can be performed 1 The landmarking process here is similar to landmarking before matching To set the first landmark select the first spot to be set as a landmark on the master image If the spot selected on the master image is incorrectly matched click on Break Match Then select the spot on the match image that corresponds to the correct match on the master image If this spot is also incorrectly matched click on Break Match Now select the two corresponding spots so that both spots become magenta and click the Add Match button Note It is only necessary
13. Cancel ero Help Changing the default radius of picking references allows the user to define the reference marker for Ettan Spot Picker The default picking head diameter is used to define the size of the automated picking head For example Ettan Spot Picker manual recommends a 2 mm picking head diameter Therefore with a 100 micron resolution image this translates to a 20 pixel picking head diameter the default value Selecting the Auto center selected spots check box results the image view automatically shifting so that the selected spot is in the center of the view DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 35 DIA Differential In gel Analysis Module 36 3 4 2 3 D View The 3 D View function provides a three dimensional representation of the primary and secondary images localized on the selected spot representing the raw image without filtering The representation is plotted along the X Y axes in the plane of the gel The Z axis scale is normalized between the two images based on the volume ratios facilitating direct visual comparison between the two 3 D spot images Three toolbar functions are associated with the 3 D View All these functions can also be accessed through menu pull down options View 3 D View Expands the 3 D View to fit the workspace View Area in 3 D Displays the area selected in the Image View in the 3 D View View Rotate 3 D Rotates the 3 D View re clic
14. Gel 02 Cy2 Standard gel El Gel 02 Cy3 Treated gel El Gel 02 Cy5 Control gel E Gel 03 Cy2 Standard gel E Gel 03 Cy3 Control gel E Gel 03 Cy5 Treated gel E Gel 04 Cy2 Standard gel E Gel 04 Cy3 Treated gel E E E Gel 04 Cy5 Control gel E Pick gel My Network File name Gel 01 Cy2 Standard gel bf Places Files of type Gel image files tif gel SE DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial IV Fully automated identification of differentially expressed proteins When the Load Secondary Gel Image window appears double click on the image Gel 01 Cy3 Control At this point the two images are displayed in the top left area of the screen Having loaded the images the next stage is to perform Spot Detection 13 4 2 Spot detection 1 After the images have been loaded select Process Process Gel Images Hep Process Gel Images 2 When the Process Gel Images menu option is selected the following dialog box appears Process Gel Images Algorithm Selection Co Detection algorithm Version 5 00 01 Description Co detection algorithm Performs spot detection in one or more images based on features in the image contrast Estimated no of spots 2500 Autodetect Picking references D Cancel Help 3 Enter the value 2500 for the Estimated no of spots see help file for an explanation of this value Then
15. IV Average Ratio 15 or IV Average Ratio 1 5 e I One way ANOVA value Properties for proteins in spot map IV Volume gt 1 00e 005 and lt 1 00e 008 TX co ordinate gt and lt TY co ordinate gt and lt Filter x Cancel Help Note The volume filtering criteria is applied to spot volumes on the Master gel If a preparative gel is assigned the parameters can be applied to this pick gel see tutorial III 3 The subset of spots that meet the criteria will now be assigned with a protein of interest status in the protein table Alternatively individual spots can be assigned manually by clicking on the spot of interest and clicking in the Protein of Interest check box at the bottom of the Protein Table screen T Pick f PTM p is Confirm Mw Da J Protein of Interest ee 194 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial Il Employing an internal standard to Analyze Protein Changes Oy AA Select View Properties or click on the Properties icon Go to the Protein Table tab and check the Protein of Interest 1 only box Click OK Now in the Protein Table are only those spots that are of most interest and can now be manually confirmed 11 9 2 Spot confirmation in Protein Table A spot should be confirmed when a significant difference has been studied and the user is certain that it is a real protein spot and it has been correctly matched on all gels 1 To confirm the first spot in the Protein
16. There is often very little benefit in investigating spots that do not change in expression levels Select View Properties and select the Table View tab Ensure in this window that the Decreased and Increased options as well as Picked Spots are ticked and that the Excluded box is not ticked Click OK It is useful to have the Confirmed option checked DeCyder Differential Analysis User Manual 18 1173 16 Edition AA DIA Differential In gel Analysis Module E DeCyder DIA Properties Workspace Spot Display Image View 3D View Table View Colors Included spots Similar V Decreased IV Increased 7 Excluded spots M Unconfirmed spots IV Confirmed spots IV Picked spots Cancel Apply Help 2 Only confirm spots that have been assigned with a pick status This is the recommended option if the protein filter has been applied prior to spot confirmation Select only Picked spots in the Table View tab The procedure is then identical to option 1 3 All spots Decreased Increased and Similar are manually verified this takes approximately 1 5 hours for 1000 spots and is not recommended Select the Similar Decreased and Increased options on the Table View tab Click OK The procedure is then identical to option 1 With the above options a convenient way to start is to sort the Table View data based on a spot characteristic e g Max Volume Click once on the column header entitled Max Volume Scroll to
17. Zoom Contrast Brightness Alt B Image View Histograrn View 3D View Table View All Views Properties Alt Enter Click on any of the four views available to expand that view to fill the screen After expanding all the views in turn click on View and select All Views from the menu above to return to the All views display Alternatively use the icons displayed on the main toolbar Note If after detection has been completed the Table View remains empty click on the Properties icon to bring up the properties dialog box Alternatively click on View Properties By default this will open on the Workspace tab Change to the Table View options by clicking on the Table View tab The gel images displayed in the image view can be changed using the pull down menus in the image view title bar Primary Gel 01 Cy2 Standard gel_ Secondary Gel 01 Cy3 Control Pe SA Sal Gel01 Cy2 Standard gel Gel 01 Cy3 Control gel Gel 01 Cy5 Treated oF gt d gel ATA i a El H 3 E 8 8 a E Y 8 i When using an experimental design that includes an internal standard as in this case it is conventional that the primary image view stays as the image of the internal standard whereas the secondary view displays the images of the analytical gels DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial Il Employing an internal standard to Analyze Protein Changes Oy
18. autodetect the references A DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 123 6 Batch processor E The Exclude Filter dialog box automatically appears Item 1 Exclude Filter Spot Properties Use Area of Interest Area of Interest I Area lt X dist IT Peak Height ES Y distance IT Volume lt The Exclude Filter dialog box can either be left empty in which case click OK or a set of known filter parameters can be entered followed by clicking OK The filtering values are best pre determined in the DIA using one of spot map pairs to be batch processed See section 3 5 1 for further details The Exclude Filter values stipulated are used on all subsequent gels The next dialog box allows a second gel to be entered All subsequent sets Of images are loaded in an identical manner to the first When all the images have been loaded click Cancel The batch information is then entered into the DIA batch list example below DIA Batch List BVA Batch List No Status Primary Label Secondary Label Tertiary Label Type DIA File XML File Estimated Auto Found Area of interest Ria 1 Pending Gel01 Standarc Cy2_ Gel01 Cy3 gel Cy3_ Gel01 Cy5 gel Cy5 Min Gel 1 dia Gel 1 xml 2500 Whole Image 2 Pending Gel02 Standarc Cy2 Gel02 Cy3 gel Cy3_ Gel02 Cy5 gel CyS Min Gel2dia
19. intervention The Batch Processor can be configured to analyze several gels in the DIA module exclusively Alternatively multiple gels can be processed through both modules to produce a final BVA file and subsequent pick list XML Toolbox The XML Toolbox enables the extraction of user specific data from the XML files generated in either the DIA or BVA modules This data can be saved in either text or html format enabling users to access data in DeCyder Differential Analysis Software workspaces for other applications The XML Toolbox also provides an interface for linking Ettan DIGE system data with Ettan Laboratory Workflow System DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 21 tg Introduction to DeCyder Differential Analysis Software 22 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Computer requirements and installation el 2 Computer requirements and installation setup cxe 2 1 Computer requirements e Operating System Windows XP e Minimum Processor Pentium 4 processor 1 5 GHz e 1 Gbyte RAM e Video card capable of 32 bit color e The video card driver needs to support Open GL y 1 2 or later ensure the latest compatible driver is installed e The color resolution of the PC should be set to 32 bit color e The screen resolution should be set to 1024 x 768 pixels landscape with 24 bits Z buffer preferably 32 bits e The virtual memory should be set so that
20. 17 6003 19 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA F 2 Related documentation Document Code no Ettan DIGE system User Manual 18 1173 17 Ettan DIGE Quick Protocols 18 1164 41 Typhoon Instrument Guide 63002831 Ettan 2D MS and LWS software Laboratory 18 1167 27 Guide Ettan 2D MS and LWS software Laboratory 18 1170 99 Guide Appendices Ettan Spot Handling Workstation 2 01 18 1163 89 LWS 1 0 User Manual Ettan MALDI ToF Pro User Manual 18 1144 01 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 273 274 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Appendix G Glossary G 1 DeCyder Differential Analysis Software glossary 25D value 2 standard deviation of the spot ratio distribution 95 of the spots lie within this ratio for normally distributed data Abundance The relative volume among all spots representing a particular protein in a BVA data set The weakest spot is taken as 1 00 and the others are displayed relative to this ANOVA ANalysis Of VAriance is a family of methods used to perform statistical analysis on experimental results ANOVA 1 way One Way ANOVA test assigns statistical significance to differences in standardized protein abundance between experimental groups ANOVA 2 way Two Way ANOVA test assigns statistical significance to both separate and mutual effects of two experimental condit
21. 4 3 Creating and opening workspaces Bwa From the Desktop or Start button menu select the DeCyder Differential 2 Analysis Software BVA icon to open the module 4 3 1 Creating workspaces DeCyder Differential Analysis Software BVA requires XML files generated in the DIA section 3 7 together with the original scanned image files to create a new BVA workspace E To load XML files in BVA select File Create Workspace in the DeCyder Differential Analysis Software BVA window or select the Create workspace icon DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 53 ED BVA Biological Variation Analysis Module AA Locate the XML files and select the files for opening by holding down 54 Qe the Control key and clicking on the required files Click Open Load DIA Result on XML format Tutorial Il A My Recent Documents G Desktop My Documents My Computer My Network File name GelOS amt Gel 01m Gel 02x Ge103 z Open Places Files of type XML Result files xml y Cancel Note The XML files contain the necessary information to open the associated image files automatically providing the image and XML files are in the same relative file path when processed in the DIA module If this is not the case a message box will appear giving the option to locate the image file s Click Yes and browse to locate and select the corresponding image files The newl
22. 50 60 A M P 1 1e 020 960 251 50 60 A M 0 078 0 67 0 93 961 228 50 60 A M P 0 0034 0 34 0 50 962 220 50 60 A M P 0 83 0 57 0 64 963 214 50 60 A M 0 00013 0 012 964 207 50 60 A M 4 0e 006 0 0016 965 204 50 60 A M P 0 15 0 37 0 60 966 202 50 60 A M P 1 1e 006 0 00030 968 157 50 60 A M 0 35 0 53 0 51 969 156 50 60 A M 0 79 0 73 0 67 970 148 50 60 A M P 0 79 0 87 0 77 971 146 50 60 A M 0 21 0 14 0 87 972 144 50 60 A M 0 20 0 59 0 99 973 119 50 60 A M 6 9e 005 0 25 0 10 974 114 50 60 A M P 0 54 0 70 0 77 AMP 10 Fig D 4 Statistical outcome for protein 162 The 2 ANOVA Strain and 2 ANOVA time values are statistically significant for protein 162 Therefore there are strain specific changes in expression of this protein i e one strain consistently has higher expression of this protein Furthermore the expression of this protein increases significantly in both strains over time However there is no significant interaction between strain and time of incubation since the 2 ANOVA interact is not significant DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 265 266 Graph Yiew Master No 721 Protein ID Tryp Inhibitor Standardised Abundance Protein Table T test and Av Ratio 8 1 34 60 A M 439 728 60 60 A M P 0 97 0 20 0 88 440 727 22 60 A M P 0 031 0 021 441 726 26 60 A M 0 046 0 024 442 725 20 60 A M 443 724 54 60 A
23. AA 4 Spot quantitation expressed as a volume normalized against the internal standard is calculated in the DIA module then displayed in the Table View under the Volume Ratio column This column is automatically amended when selecting between analytical gels in the secondary gel view 11 4 4 Exporting spot map data The data associated with the spot boundaries and spot quantitation are employed by the BVA module to perform inter gel matching and further statistical analysis This data is therefore exported from the DIA module in an XML format 1 Select File Export Spot Maps File i View Process Help Create Workspace Ctrl N Open Workspace Ctrl 0 Save Workspace Ctrl S Save Workspace As Print Ctrl P Print Preview Export Result Table Export Spot Maps 2 Browse to locate the folder entitled Tutorial Il in the Tutorial data files folder Name the exported file Gel 01 then click Save Export Spot Maps Save in C Tutorial Il cf El 2 Gel 02 xml My Recent 2 Gel 03 xml Documents e Gel 04 xml D My Network File name Gel O1 xml Places Save as type XML files xml 3 The remaining gels must be subjected to similar analyses using the DIA module For the purposes of this tutorial the gels have been prepared and placed in the Tutorial II file ready for analysis in the BVA module DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 179 ED
24. ANOVA Dose 2 ANOWA Time 2 ANOVA In 16 230 Unconfirmed 52 60 A M P ASer 1 35 17 1071 Unconfirmed 44 60 A M 1 93 18 652 Unconfirmed 38 60 A M 0 0011 1 37 19 1031 Unconfirmed 44 60 A M 0 0015 1 97 21 801 Unconfirmed 52 60 A M 0 0019 1 36 22 207 Unconfirmed 50 60 A M 0 0021 1 42 23 638 Unconfirmed 38 60 A M P 0 0021 1 10 24 430 Unconfirmed 46 60 A M P 0 0022 1 18 25 525 Unconfirmed 50 60 A M 0 0022 1 06 26 237 Unconfirmed 42 60 A M P 0 0025 1 62 27 728 Unconfirmed 60 60 A M P 0 0026 1 10 28 1078 Unconfirmed 38 60 A M 0 0026 1 32 29 202 Unconfirmed 50 60 A M P 0 0027 1 88 30 592 Unconfirmed 54 60 A M P 0 0028 1 10 31 765 Unconfirmed 36 60 A M 0 0028 1 22 32 1089 Unconfirmed 26 60 A M 0 0028 1 24 33 894 Unconfirmed 54 60 A M 0 0035 1 08 34 514 Unconfirmed 48 60 A M P 0 0040 1 09 35 597 Unconfirmed 58 60 A M P 0 0040 1 09 36 576 Unconfirmed 58 60 A M P 0 0046 1 13 37 715 Unconfirmed 38 60 A M 0 0046 1 29 38 730 Unconfirmed 38 60 A M 0 0046 1 48 39 391 Unconfirmed 38 60 A M 0 0048 1 28 40 541 Unconfirmed 48 60 A M 0 0052 1 14 41 223 Unconfirmed 40 60 A M 0 0054 1 46 42 624 Unconfirmed 40 60 A M P 0 0055 1 16 43 115 Unconfirmed 30 60 A M 0 0057 1 34 44 1020 Unconfirmed 46 60 A M 0 0057 1 14 45 203 Unconfirmed 56 60 A M P 0 0059 1 76 46 625 Unconfirmed 60 60 A M P 0 0060 1 07 47 860 Unconfirmed 50 60 A M P 0 0060 1 15 48 734 Unconfirmed 60
25. Apply Help Use the Zoom in function to make the annotations more legible lt DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 93 ED BVA Biological Variation Analysis Module 4 11 User defined protein labelling 4 11 1 Confirmation Protein confirmation allows the marking of proteins for user reference purposes e g for visually checked proteins No part of the analysis in DeCyder Differential Analysis Software BVA demands confirmation of proteins To confirm a protein select the Confirm button in the data control panel If the selected protein is confirmed it can be unconfirmed by selecting the Unconfirm button 4 11 2 Name User defined names can be entered through the text box in the data control panel 4 11 3 Comment User defined comments can be entered through the text box in the data control panel 4 11 4 Protein of interest The Protein of Interest check box is selected for proteins that may warrant further investigation by the user PriSpot Map Protein ID Protein AC Name Comment i CT Pick I PTM Confirm di q T Eee Protein spots possessing a post translational modification PTM identified by methods outlined in Ettan DIGE application notes e g application note 18 1170 83 AA obtainable on the Amersham web site can be denoted by checking the PTM check box 4 12 Database linking DeCyder Differential Analysis Software provides the user with functions that enabl
26. Batch Processor can be used This automatically runs a series of sequential DIA analyses with no need for user intervention and can be set up to then automatically match the individual spot maps perform statistical analysis then generate a pick list in the BVA module 13 5 1 Setting up the batch processor The first stage of the process is to create a DIA batch list by defining which gel images are to be processed and in what order The Primary Image in every case is the specific standard image for that gel and the secondary and tertiary images are the control and treated sample images the order is arbitrary 1 Double click on the DeCyder Differential Analysis Software Batch Processor icon on the desktop to open the Batch Processor interface 2 Select File New Batch In the dialog box that now appears browse to locate the folder entitled Tutorial IV in the Tutorial data files folder 3 Select the image Gel 01 Cy2 Standard in the left hand panel then click the Primary gt button Repeat the procedure for the Gel 01 Cy3 Control and Gel 01 Cy5 Treated using the Secondary gt and Tertiary gt buttons respectively Click OK Select Images Select Gel 01 Cy2 Standard gel Gel 01 Cy3 Control gel SCA Gel 02 Cy2 Standard gel ER Firman Gel 02 Cy3 Treated gel Y Tutorial images v5 ae i gs SEIA CJ Temp Tutorial data files Gel 03 Cy3 Control gel Gel 03 Cy5 Treated gel Gel 04 Cy2 Standard gel Gel 04 Cy3 Treated gel Gel 04 Cy5
27. Batch Processor is therefore a means of automation and does not introduce further processing and analysis to the spot map data All the concepts for processing the gel images have been discussed in the DIA and BVA sections of the manual and are not further elaborated upon in this section References to the appropriate sections are made where necessary 6 2 Batch list creation Double click the DeCyder Differential Analysis Software Batch Processor icon on the desktop to open the Batch Processor The creation of the batch list can be broadly sub divided into two activities e DIA batch list set up Loading the spot map pairs for co detection in the DIA module e BVA batch list set up Stipulating spot map attributes setting up the statistical analysis and subsequent pick list in the BVA module DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 121 6 Batch processor E 621 Setting up the DIA Batch List When setting up a DIA batch list each gel is processed sequentially To create a batch list select File New Batch Select Images Select Gel 01 Cy2 Standard Gel 01 Cy3 Control gel Gel 01 Cy5 Treated gel Gel 02 Cy2 Standard gel a a e pa 3 Tutorial images v5 el 5 Control gel i Gel 03 Cy2 Standard gel Tutoria data fis Gel 03 Cy3 Control gel Gel 03 Cy5 Treated gel Gel 04 Cy2 Standard gel Gel 04 Cy3 Treated gel Gel 04 Cy5 Control gel Primary gt Gel 01 Cy2 Standard gel E Gel 01 Cy3 Cont
28. Control gel Dz Primary gt Gel 01 Cy2 Standard gel Secondary gt Gel 01 Cy3 Control gel Tertiary gt Gel 01 Cy5 Treated gel 4 In the Result Files section of the box that now appears ensure that the DIA and the XML file names end in the number one by typing 1 as indicated above as this enables the subsequent files to be 226 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial IV Fully automated identification of differentially expressed proteins named automatically with an incrementing number It is recommended that both files are named Gel 1 Click OK Item 1 Gel Image Information and Result Files Gel Image labels and type Primary Gel 01 Cy2 Standard gel Secondary Gel 01 Cy3 Control gel Tertiary Gel 01 Cy5 Treated gel Chemistry type Result files DIA filenames XML filename TE Note Ifthe name does not end in a 1 then you will be given the opportunity to name the DIA and XML filenames for each batch item The DIA results are generated in two different formats e A DIA file which contains the images and raw data information for each pair analysis e An XML file which contains the raw data file that is subsequently exported to the BVA Biological Variance Analysis module 5 Enter the value 2500 for the estimated number of spots and ensure that the Auotdetected Pick Reference markers check box is deselected and the Include in BVA batch list ch
29. DIA module for preliminary investigation of protein changes 10 Before going any further it is important to become acquainted with the main tool bar DSH A 8 R aea R SN Create Workspace What s this Open Workspace Print Save Workspace iii Properties ProcessGels All Views Protein Filter Table View Exclude Filter 3D View Areain3D Histogram View Rotate 3D Image View Zoom In Contrast and Brightness Zoom Out Fit to Window The user interface is divided into four main windows the Image View the Histogram View the 3 D View and the Table View In addition the Data Control Panel at the bottom of the screen allows user defined attributes to the spot selected in the four views F Decyder DIA Control Treated dia File Edt View Process Help Ce Primary Gel 02 Control Cy3 v al 3 856e 005 Rx 1 8 R lE Spot No 2309 Position 1143 1106 Pick pos amalas Vi Pi A Number of Spats Status Log Volume Ratio Spot No Histogram selections Scatter parameter Max Volur Threshold mode 2 model S Threshold 2 41551 25D 2 32553 Spot statistics Decreased Similar Increased Detected Included Excluded Picked Abundance Excluded Volume Ratio 116 4 8 2235 91 6 90 3 7 w Histogra 2441 2441 o o Picked_ Funct Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unco
30. DIGE system see Appendix B DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 29 DIA Differential In gel Analysis Module Load Primary Gel Image Look in E Tutorial Il z e gm co Gel 05 Cy5 Treated gel Gel 01 Cy2 Standard gel E SYPRO gel Gel 01 Cy3 Control gel O a E My Recent Documents E Gel 01 Cy5 Treated gel E Gel 02 Cy2 Standard g E Gel 02 Cy3 Control gel Desktop E Gel 02 Cy5 Treated gel E Gel 03 Cy2 Standard gi E Gel 03 Cy3 Control gel E Gel 03 Cy5 Treated ge El Gel 04 Cy2 Standard g E Gel 04 Cy3 Control gel E Gel 04 Cy5 Treated gel E el 05 Cy2 Standard ge E Gel 05 Cy3 Control gel My Documents gt My Network File name Gel 01 Control Cy3 gel bd Net d oe Files of type Gel image files tif gel v Cancel Repeat for the secondary and tertiary images if required Note In an experiment in which an internal standard sample is being used the standard sample image is designated as the primary image and the analytical samples are designated as the secondary and tertiary images Once the image s have been loaded spot detection and quantitation can be performed Opening Workspaces To open previously created and saved workspaces select File Open and browse to locate the DIA file When the file is located select the file and click Open or double click on the DIA file 30 DeCyder Differential Analysis User Manua
31. Descr Condition Condition2 Sample 1D Comment Gel 01 Cy2 Stanc Min Cy2 A Standard Gel 01 Cy3 Contr Min Cy3 Control Gel 01 Cy5 Treat Min _ Cy5 Treated Gel 02 Cy2 Stanc Min _ Cy2 Standard Gel 02 Cy3 Treat Min_ Cy3 Treated Gel 02 Cy5 Contr Min_ Cy5 Control Gel 03 Cy2 Stanc Min Cy2 Standard Gel 03 Cy3 Contr Min_ Cy3 Control Gel 03 Cy5 Treat Min Cy5 Treated Gel 04 Cy2 Stanc Min Cy2 Standard Gel 04 Cy3 Treat Min Cy3 Treated Gel D4 Cy5 Contr Min Cy5 Control Pick gel Min Unas Unassigned Ea Fe eee The Batch Processor workspace is now completed and ready to be run Note All items in the Batch Processor workspace can be edited by double clicking the relevant cell in the DIA and BVA batch list or selecting the relevant dialog window button on the top of the screen 234 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial IV Fully automated identification of differentially expressed proteins 13 5 7 Running the batch processor Select Process Run batch A prompt appears to request automatic setting of the Master the spot map that all gels are matched with Select Yes and the gel with largest number of detected spots will be assigned as the Master spot map Click OK in the next dialog box if you wish to start processing immediately Alternatively
32. Edition AA 143 ED LWS Integration 144 8 4 Enter a gel prepared for spot picking within Ettan DIGE system into Ettan LWS A preparative gel or an analytical gel with CyDye DIGE Fluor dye labelled proteins can be used as a picking gel For details of the methods for the different routes to picking refer to chapter 5 The workflow for the gel should be set up the normal way in Ettan LWS The only differences are within sample attributes template and sample definition 1 Set up a sample attribute template as in 8 3 step 1 The samples need to be defined in Scierra web 2 In Scierra web select project name and Define Samples 3 Scan or type sample ID choose Sample Type Protein Sample Template the user defined template created in step 1 and Container Gel 2D and enter values of the sample attributes An example is shown in Fig 8 5 This extra information helps the user to keep track of which samples are in each gel 4 Select Glass backing fill in backing thickness 3 2 5 Browse for the image file and the converted evaluation file converted according to section 8 5 to be used Note Image files to be included in the sample definition must have a file name of less than 45 characters otherwise a work request for that sample cannot be generated and an error message will be shown Note If samples generated within Ettan DIGE system are entered as a gel it will only be possible to track the samples in the spot handlin
33. Export Spot Maps so that it can be loaded into the BVA module for matching against a preparative gel Both the preparative gel and analytical gel XML files are loaded into the BVA module see section 4 3 1 and matched Landmarking may be required to accurately match preparative gels see section 4 6 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 111 ER Spot picking 112 One of the analytical spot map must be assigned as a template spot map in the Spot Map Table mode of the BVA module by selecting the Template check box in the data control panel see section 4 5 Function for Spot Map SYPRO gel Analysis A Master M W Template T Pick P Spots assigned as protein of interest are denoted by the presence of the letter I in the Function column of the Protein Table and have the Protein of Interest check box selected 5 4 2 Identifying proteins of interest using the BVA module The simplest means to assign POI status is to manually choose spots then select the Protein of Interest check box when in Spot Map Table mode pl CT Pic PTM confirm Mw DR Protein of Interest Alternatively identification of proteins for picking can be based on the statistical information generated from a BVA workspace containing several spot maps that have been subjected to statistical analysis within the BVA module Proteins can be selected using various criteria via the protein filter Click on the Protein Fi
34. Gel 2 xml 2500 i A Whole Image 3 Pending _ Gel03 Standarc Cy2 Gel03 Cy3 gel Cy3_ Gel03 Cy5 gel Cy5 Min Gel3 dia Gel 3 xml 2500 S 04 0P 0W 0 Whole Image 4 Pending Gel04 Standarc Cy2 Gel04 Cy3 gel Cy3_ Gel04 Cy5 gel Cy5 Min Gel4 dia Gel 4 xml 2500 0 4 0 P 0 V 0 Whole Image 5 Pending Gel05 Standarc Cy2 Gel05 Cy3 gel Cy3_ Gel05 Cy5 gel Cy5 Min Gel5 dia Gel 5 xml 2500 0 4 0 P 0 V 0 Whole Image 6 Pending Gel06 Standarc Cy2 Gel06 Cy3 gel Cy3 Gel06 Cy5 gel Cy5 Min Gel 6 dia Gel 6 xml 2500 S 04 0P 0V 0 Whole Image 7 Pending Gel07 Standarc Cy2_ Gel07 Cy3 gel Cy3 Gel0 Cy5 gel CyS Min_ Gel 7 dia Gel 7 xml 2500 0 4 0 P 0 V 0 Whole Image 8 Pending Gel08 Standarc Cy2 Gel08 Cy3 gel Cy3_ Gel08 Cy5 gel CyS Min Gel8 dia Gel 8 xml 2500 5 04 0P 0V 0 Whole Image 9 Pending Gel09 Standarc Cy2 Gel09 Cy3 gel Cy3_ Gel09 Cy5 gel Cy5 Min Gel 9 dia Gel 9 xml 2500 5 04 0P 0V 0 Whole Image 10 Pending _ Gel10 Standarc Cy2 Gel10 Cy3 gel Cy3 GellO Cy5 gel CyS Min Gel10 dia Gel 10 xml 2500 Whole Image 11 Pending Gell1 Standarc Cy2_ Gell1 Cy3 gel Cy3 Gell1 Cy5 gel CyS Min Gel 11 dia Gel 11 xml 2500 Whole Image 12 Pending _ Geli2 Standarc Cy2_ Gell2 Cy3 gel Cy3_ Gell2Cy5 gel CyS Min Gell2dia Gel 12 xml 2500 Whole Image 13 14 15 XA r If inclusion of a BVA batch list was selected the user is prompted to set the BVA workspace Question Set BYA workspace 124 DeCyder Differential
35. Manual 18 1173 16 Edition AA 45 DIA Differential In gel Analysis Module Protein ID 46 detected spots on the basis of these values In addition those spots outside of the area of interest will also be removed Note These excluded spots can be returned to the experiment by de selecting the check boxes in the Exclude Filter window and then re running the filter Alternatively the values in the exclude filter can be edited followed by re running the filter Excluding spots only removes them from the analysis not from the workspace Manual spot exclusion Non proteinaceous spots that are not removed by the exclusion filter can be manually removed from the data set by highlighting the spot then selecting the Exclude check box at the bottom of the screen Pick p z Comment Confirm Protein of Interest Select Process Re Normalize to perform the normalization process again with the manually excluded spots removed from the calculation Note Re normalization is automatically performed when the Exclude Filter is used 3 5 2 Spot confirmation Remaining non excluded spots can be manually confirmed for the purposes of visually verifying each spot Three options are available during spot confirmation 1 Only confirm spots that are increased or decreased in their abundance This method is relatively rapid since the increased and decreased spots are automatically identified by setting the threshold mode see section 3 4 4
36. Table Appearance Table Image View 3D View GraphView Database Colors Include in Image View IV Spots present in table only Spot Features Signature I Annotation vi la Show Pick Protein IV Match vectors when Match Table is displayed Scrolling and Auto Center Settings Link image views when scrolling IV Auto center selected spots Pickince cepts Picking references and pick locations Defouk SEE of picking references 5 100 Default diameter of picker head 5 50 Secondary Image View Default radius of picking references 5 100 Default diameter of picker head 5 50 Cares T DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 117 ER Spot picking The pick locations are displayed as a yellow by default transparent cylinder and a yellow circle in the 3 D View and image view respectively when a picked spot on the pick gel is selected The pick locations can be edited by zooming in on the selected spot in the image view then selecting Edit Edit Pick Locations Place the hand shaped cursor that appears over the centre of the pick location that requires editing in the image view then drag the pick circle to the desired location This can be repeated for pick locations that require editing Select Edit Edit Pick Locations to exit this mode O To return all pick locations to the initial position of centre of mass select Edit Restore Default Pick Locations Note Editing of pick locations can
37. There are therefore three sets of hypothesis with the Two Way ANOVA The null hypotheses for each of the sets are given below The population means of the first condition are equal This is like the One Way ANOVA for condition 1 exclusively The population means of the second condition are equal This is like the One Way ANOVA for condition 2 exclusively There is no interaction between the two factors A significant ANOVA Interaction value indicates that the two factors affect each other due to synergy or interference Examples of such effects are illustrated on next page DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 85 ED BVA Biological Variation Analysis Module 86 1 2 o Oo y A B A B No significant variation P lt 0 001 P not significant A B A B P not significant P lt 0 001 P lt 0 001 P lt 0 001 P not significant Treated O Control A B P lt 0 001 P lt 0 001 P lt 0 001 Graphical examples of Two Way ANOVA analyses Graphs illustrate changes in protein abundance y axis for a two condition experiment Condition 1 x axis represents two temperatures A and B condition 2 red and yellow circles represents drug treated and control samples Each condition is in triplicate hence there are four experimental groups with 3 samples in each group Conditions 1 and 2 are used to link groups together based on one common factor i e gr
38. View Process Help D d ist Rae 2922900003018 Primary Gel 02 Control Cys Histogram selections Scatter parameter Max Volur_ Threshold mode 2 model S Threshold 241551 25D 2 32553 Spot statistics Decreased 116 4 8 Similar 2235 91 6 Increased 90 37 a w Histogram View Detected 2441 Included 2441 Number of Spots SURYA xew Status Excluded Unconfirmed Unconfirmed lume 3 856e 005 Spot No 2309 Vi Unconfirmed Position 1143 1106 Peak Height 882 Position 1143 1106P pane Pick pos Area 1419 Pick pos As T Pick T PIM Protein ID y Confirm T Protein of Interest I Exclude Focus Secondary 3D View NUM The user interface is divided into four main windows that show the Image View the Histogram View the 3 D View and the Table View The four views are linked Clicking on any spot on the Image View DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 177 ED 1 Il Employing an internal standard to Analyze Protein Changes 178 highlights the spot in magenta in the Histogram View The spot is also represented three dimensionally and is highlighted in the Table View Each of the four views can be expanded to fill the whole screen To do this click on the View button and the menu below is displayed View Process Help Area in 3D Ctrl A Rotate 3D Ctr R
39. View properties window The size of area displayed in the 3 D View can be altered by entering a positive integer between 3 and 80 DeCyder DIA Properties Workspace Spot Display Image View 3D View Table View Colors Spot margin for displayed spots 1d SY DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 37 DIA Differential In gel Analysis Module A 34 3 Table View Displays data associated with selected co detected spots in a tabulated format The data within the table can be sorted into ascending or descending order by clicking on the column headers of the table Status spot No Abundance Excluded volume Ratio Picked Max Slope Area Max Peak Clicking on the Table View icon or selecting View Table View expands the EJ Table View to fit the workspace The following information on co detected spots is contained within the Table View The spots displayed in the table can be adjusted using the Table View Properties Tab see section 3 5 1 Status Indicates whether a spot has been confirmed by the user see section 3 5 1 Spot No Spot reference number unique to a spot pair on a set of images Abundance Decreased Similar or Increased depending on thresholds set in DIA Histogram View Excluded Spot assigned by user or Exclude Filter for removal from analysis set An excluded spot is never completely removed from the workspace and can be recovered by the use
40. also be performed in the DIA module To display only picked spots in the image view select Picking references and pick locations check box in the Spot Display tab of the DIA module Properties dialog window Editing pick locations is then performed in an identical manner to the BVA module 118 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Spot picking ER 57 Generating a Pick List Pick lists can be generated for either Ettan Spot Picker or Ettan Spot Handling Workstation in both the BVA and DIA modules Select File Export Picking List From Pick Spot Map in the BVA module to generate a pick list for the preparative gel A pick list is generated in the DIA module by selecting File Export Picking List The pick list can then be saved as either a text file or an XML file by selecting the appropriate file extension to the file name txt or xml The text and XML file are used for Ettan Spot Picker or Ettan Spot Handling Workstation respectively DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 119 EN Spot picking 120 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Batch processor 6 6 Batch processor Batch A 6 1 Overview The Batch Processor links both the DeCyder Differential Analysis Software DIA and BVA modules to perform all stages of the 2 D DIGE analysis process Once the Batch Processor has been set up the gels are processed sequentially without user intervention The
41. an xml file that can be imported into the BVA module Data that can be imported from Ettan LWS to DeCyder BVA are e Protein ID If multiple protein candidates are included in the LWS report for the different picked spots the protein Id s of the different candidates are all appended in a single string separated by semi colons e Protein Name If multiple protein candidates are included in the LWS report for the different picked spots the protein names of the different candidates are all appended in a single string separated by semi colons e Molecular Weight MW The MW value of the first protein candidate in the list is added to Ettan Protein Identification data DeCyder Differential Analysis User Manual 18 1173 16 Edition AA LWS Integration 8 A Isoelectric Point PI The pl value of the first protein candidate in the list is added to Ettan Protein Identification data 8 6 1 Preparations in Ettan LWS before XML conversion After MALDI mass spectrometry is completed the Detected proteins in gel report can be generated 1 In Scierra Web select Tools Reports and generate the standard report Detected proteins in gel as described in Ettan 2D MS and LWS Software Laboratory Guide Note Select how much information to import into DeCyder BVA by altering the maximum rank and by including sub rankings or not The report will appear on screen Detected proteins in gel Showing which proteins are repre
42. at least two members in each are required The experimental design should also be set up so that the following criteria are applied e Condition 1 The experimental groups should have both condition values filled in so that there are at least two groups with different condition 1 values and the same condition 2 value e Condition 2 The experimental groups should have both condition values filled in so that there are at least two groups with different condition 2 values and the same condition 1 value Additional requirement for paired Two Way ANOVA e At least two different sample IDs in each group spot maps with the same condition 1 and condition 2 value DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 89 ED BVA Biological Variation Analysis Module 90 For further information on experimental design and set up examples see Appendix D Experimental design and set up examples 4 8 9 Further statistical analyses The data generated within DeCyder Differential Analysis Software can be exported as an XML file and then extracted using the XML toolbox enabling post processing of several parameters The user can therefore apply their own statistical analysis algorithms to normalized and pre normalized data to suit their own specific requirements 4 9 Protein Filter The protein filter is a tool that allows the selection of proteins based on various user defined criteria To open the protein filter select Proces
43. be carried out on an image it must first be acquired and stored within the computer in a suitable form Image acquisition is a critical step and all primary data should ideally be stored exactly as it is recorded by the imaging device without significant data compression as this may affect the accuracy of the recording It should also be of the highest quality required for image resolution in the particular application Acquisition can be achieved using a variety of scanners or digital imagers These are usually based on PhotoMultipier Tubes PMTs or Charge Coupled Devices CCD A PMT is an electro optic device that converts light energy into electrical current and amplifies the current whilst a CCD is a silicon based integrated circuit consisting of a dense matrix of photodiodes that operate by converting light energy in the form of photons into an electric charge B 2 The digital image The most practical way of storing an image as digital data is to divide the image into a grid of very small regions called picture elements or pixels In the computer this digital grid or bitmap represents the image Each pixel is identified by its position in the grid as referenced by its row x and column y number Each pixel has a different color or gray scale value and together they form a representation of the image DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 243 B 3 AA The images below show the digita
44. boxes to display only proteins of interest or pick assigned proteins in the Image View and the Table View Both check boxes can be selected if there are spots selected as proteins of interest and pick status Table Column Order and Visibility The selected column titles are displayed in the Table View Clicking and dragging the column titles in the list sets the order of the columns in the Table View Clicking Default restores the original settings 100 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN E 4132 Appearance Table The table below describes the information contained in the Appearance Table No Spot map number Image Gel image file name Type Indicates the dye chemistry used Label Indicates the CyDye DIGE Fluor minimal dye label Function Spot map function e g Master Analysis Pick and Template Abundance Relative volume of all spots representing a particular protein in a BVA data set used when no internal standard is present Std Abundance Protein volume calculated relative to internal standard Volume Spot pixel volume expressed background subtraction Peak Height Largest pixel value within the spot boundary expressed background subtraction Group Assigned spot map group Group Description Description of spot map group Condition 1 Condition 1 numerical value Condition 2 Condition 2 numerica
45. check boxes the various categories of spots can be selectively displayed Deselecting all the check boxes results in the Table View being blank Similarly spots can be selectively displayed in the image view using the Spot Display tab The Table View tab works on OR logic i e a spot only needs to conform to one criterion to be shown in the table DeCyder DIA Properties 3D View TableView Colors Workspace Spot Display Image View Included spots IV Similar IV Decreased IV Increased I Excluded spots IV Picking references and pick locations Picked spots DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial Using DIA module for preliminary investigation of protein changes 10 Selecting different protein spots on the images reveals that some of the spots that have been detected are in fact either dust particles or artifacts from the gel To remove these artifacts an Exclude filter must be used on the images 10 4 3 Assigning an area of interest Due to gel heterogeneity at the edges of the images there are artifacts that need to be removed These can be removed by setting an area of interest This function only works with the filter If an area of interest was set before detection all the spots on the image will still be detected even those outside the area of interest 1 Click on the Fit to window icon on the tool bar to fit the images to the Image View 2 Click on the I
46. connecting Mean Values IV Standards IV Legend Y axis Settings Automatic C Manual Manual with automatic increase Max Min Cancel Apply Help Parameter visualization Allows the user to define how data is displayed on the graph by designating the parameters displayed on the X and Y axis of the graph DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 71 ED BVA Biological Variation Analysis Module 72 The X axis options allow the user to display data according to group conditions or fluor type The sequence of the groups on the X axis are determined by the order of group folders in the Experimental Design view within the Spot Map Table mode This can be changed by dragging and dropping the folder into the desired order within the Experimental Design view The Y axis options allow the user to display either the abundance log of the abundance standardized abundance or log standardized abundance However the statistical functions within DeCyder Differential Analysis Software only utilize log standardized abundance Therefore the graphical representation of this parametric value only reflect the data points used in the statistical analyses Dashed line options allow the user to display dashed lines on the scatter plot linking data points by the specified association Samples either derived from the same gel or possessing the same sample ID can therefore be easily identified in the graph view Inclu
47. created in step 1 and Container tube and enter values of the sample attributes An example is shown in Fig 8 4 Note The extra information Sample attribute values helps the user to keep track of which samples are in each gel Sample Definition Sample Definition Import Sample Definition Ettan 2D MS Project Selection ID tn413 E Add to Project Project Name M E Coli protein mapping Sample Type Protein go ae a AE A UU Sample Template DIGE zl Comments Container Tube fea See he SE Sample Attributes Name Value Concentration mg ml Name Volume ul Cy2 S1 pool Cy3 si control 1 cys si treated 2 Protein labelling 50 pg 400 pmol Experiment E coli benzoic stu Finish Activity Clear Window Close Window Fig 8 4 Example of Sample Definition and entered values for the attributes when entering a sample generated within Ettan DIGE system as a protein tube Methods Normally the standard methods included in Ettan LWS software and in Ettan MALDI ToF Pro can be used If non standard methods are to be used these methods have to be added e In Management Workbench System Method editor add staining method e In Scierra LWS select Spot Handling from the Workflow Graph In Tools Spot Handling Method Management Method Overview add Spot Handling methods e In Ettan MALDI Control Methods add MALDI methods DeCyder Differential Analysis User Manual 18 1173 16
48. detection and quantitation on a set of images from the same gel BVA Biological Variation Analysis Matches multiple images from different gels to provide statistical data on differential protein expression levels between multiple groups Batch Processor Fully automated image detection and matching of multiple gels without user interaction XML Toolbox Extracts user specific data facilitating automatic report generation DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 19 ED Introduction to DeCyder Differential Analysis Software 2 D DIGE gel 20 Batch Processor XML file XML file Image Image gt em Me Fora ne BVA Mea Experienta conditions Fig 1 4 Structure of DeCyder Differential Analysis Software DIA module A set of images each saved as either 16 bit TIFF or customized GEL file from a single gel are loaded then processed simultaneously The DIA algorithms perform spot detection on a combined image derived from all loaded images The protein spot quantitation is calculated and expressed as a volume ratio to the internal standard The results can be saved as a DIA file which can be re opened in the DIA module This pick list is created from data generated in a single DIA module analysis i e an experiment based on a single gel Alternatively the generated results can be saved in an XML format or opened in the XML Toolbox module in order
49. for use by other applications A Result Microsoft Internet Explorer File Edit View Favorites Tools Help Ey Bac P x E JO Search She Favorites O media amp B ES E El about blank All proteins file G Firman Ghouze 2D DIGE Manual Tutorial images Gare as Jet DIA spot no Volume ratio Volume ratio Volume ratio Commer 4479 6 1688 3 9337 3281 9790 4613 6413 9196 7674 5363 8747 3286 4094 5600 6423 2707 0576 4013 2419 1149 5460 6295 6789 5179 6503 2187 0386 3124 9859 2111 3829 9880 2980 8233 5439 3137 AOniInaananw b b OO bh b IN nn Y 4 My Computer 7 4 Tag definitions 7 4 1 BVA parameters Experimental data Condition 1 Numerical condition 1 assigned to the spot map group Condition 2 Numerical condition 2 assigned to the spot map group DIA experiment File name of the corresponding DIA workspace Dye chemistry Indicates whether minimal or saturation labelling was used Dye label Indicates the fluor used to label the protein Group Description Group Name of the experimental group that the spot map has been assigned to Image name Gel image filename DeCyder Differential Analysis User Manual 18 1173 16 Edition AA XML Toolbox No of included spots Number of spots included in the data set No of matched spots Number of spots matched to the master image Sample ID Numerical identifier assigned to spot maps for paired statistical an
50. gels seen as perfect circles on the images act as these reference points The position of the reference markers are automatically detected during the spot detection process However it is advisable to review the position of the reference markers and edit them if necessary 1 Zoom into the area of this reference marker by holding down the left mouse click to draw a rectangular area around the marker 2 The size of the target can be altered by clicking on the properties icon and selecting the Image View tab A value of approximately 30 should be entered in the Primary Image View box of the picking references area of the window DeCyder BYA Properties Spot Map Table Match Table Protein Table Appearance Table Image View 3D View GraphView Database Colours Include in Image View IV Spots present in table only Spot Features IV Signature I Annotation m a z IV Match vectors when Match Table is displayed Scrolling and Auto Center Settings IV Link image views when scrolling IV Auto center selected spots Picking Reference Data IV Picking references and pick locations Master Image View Default radius of picking references 5 100 fso Default diameter of picker head 5 50 20 Primary Image View Default radius of picking references 5 100 fso Default diameter of picker head 5 50 20 Cancel DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 211 ED 1 Ill
51. igi alumn mi Secondary Image View Default radius of picking references 5 100 Default diameter of picker head 5 50 Cone Coca amm Her Restore to default order and selection Default The Protein Table and the image view will then display only the proteins that have been assigned with a pick status Using the Zoom in icon the individual spots selected for picking can be seen Each of the protein spots have a black dot within the spot boundary denoting the centre of mass of the spot and hence the centre at which a picking head will pick from the gel The centre of mass represents the optimal picking location for a vast majority of spots However it may be advantageous to edit the pick location when two spots are in very close proximity in order to minimize the possibility of cross contamination The picking location can therefore be edited in these cases DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Spot picking ea The picking location must first be displayed in both the image view and 3 D View when in Protein Table mode The pick location is only displayed on the spot map assigned as pick providing it has the picking references defined To display the picking locations ensure that the Picking references and pick locations check box is selected in the Image View tab of the Properties dialog window DeCyder BYA Properties Spot Map Table Match Table Protein
52. image is stipulated the internal standard spot map if present with greatest number of detected spots is automatically assigned as the master image when running the batch list Once all spot maps attributes have been defined the Protein Statistics dialog box automatically appears This allows the user to perform statistical analysis on the experiment The appropriate statistical analysis can be set up as described in section 4 8 Click OK to confirm DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 125 6 Batch processor 126 the statistical analysis entered Alternatively if no statistical analysis is required click Cancel The Protein Filter dialog box then appears automatically The filter allows either the highlighting of interesting proteins for further investigation or the direct identification of proteins for picking and subsequent generation of a pick list if a pick gel is present in the batch list see Chapter 5 Note It is recommended that the Protein Filter is used to highlight interesting proteins for further investigation These proteins can subsequently be confirmed by the user in the BVA module prior to generating a pick list see section 5 4 2 6 3 Editing the batch list 6 3 1 DIA batch list To edit the DIA batch list select View DIA Batch List to ensure the DIA batch list is displayed Adding To add further spot map pairs to a current DIA batch list select the next empty row in the batch
53. image to be assigned as master using the left mouse click Tick the box entitled Master at the bottom of the table in the area entitled function for spot map However the Master should not be changed here or all the matching will have to be repeated 7 To add the SYPRO stained preparative gel spot map to the BVA workspace select File Import Spot Map s and double click on the XML file exported from the DIA workspace of the Preparative gel Import Spot Maps Look in O Tutorial IV gt misc My Recent Documents Desktop A My Documents My Computer HU NORGE File name Pick xml laces Files of type XML Result files xml Cancel Once loaded the SYPRO gel spot map can be assigned as a Pick gel ensuring that the eventual pick list will based on the spot co ordinates of this SYPRO gel image Select the SYPRO spot map in the table view then select the Pick check box in the data control panel De select the default Analysis check box Function for Spot Map SYPRO gel I Analysis A TT Master M I Template T IV Pick P 210 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial III Processing the Preparative Gel and Generating a Pick List 12 12 4 2 Editing picking reference markers The software that controls the picking robot requires two reference points in order to locate the spots for picking from the preparative gel The reference markers placed on the preparative
54. limited license to use the CyDye fluors for internal research and development but not for any commercial purposes A license to use the CyDye fluors for commercial purposes is subject to a separate license agreement with Amersham Biosciences Amersham Biosciences has patent applications pending relating to its DeCyder software technology European patent application number EP1 234 280 Office Addresses Amersham Biosciences AB Bj rkgatan 30 SE 751 84 Uppsala Sweden Amersham Biosciences UK Limited Pollards Wood Nightingales Lane Chalfont St Giles Buckinghamshire HP8 4SP Uk Amersham Biosciences Corp 800 Centennial Avenue P O Box 1327 Piscataway N J 08855 1327 USA Amersham Biosciences Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany Amersham Biosciences KK Sanken Building 3 25 1 Hyakunincho Shinjuku ku Tokyo Japan OAmersham Biosciences AB 2003 All rights reserved Contents 1 Introduction to DeCyder Differential Analysis Software LL Intro UC recia a o torsd dn enone ly ld 11 1 2 The DeCyder Differential Analysis Software User Manual 1 1 3 Measuring differential protein abundance using Ettan DIGE System 00 a 2 1 4 Experimental Design using an Internal Standard oooooccccccnnnc 4 1 5 Integration of DeCyder Differential Analysis Software with Ettan DIGE system experimental design cccccccnnonocococoooooonnnononoss 7 1 6 Steps involved in Image Analysis us
55. list and select File Add DIA batch item Extra gels are added as described in section 6 3 2 Removing To remove an item select the item from the DIA batch list select File Remove DIA batch item then confirm that the item is to be deleted The item will then be automatically removed Editing If an incorrect image is selected while loading the DIA batch list continue loading the rest of the images then amend the desired image at the end To amend the batch list select the desired item from the DIA batch list and then select Edit Edit Item 6 3 2 BVA batch list To edit the BVA batch list select View BVA Batch List to ensure the BVA batch list is displayed Adding To add a spot map s to the BVA batch list select File Add Spot Map s Browse to locate and select the XML files corresponding to the spot map s processed in a previous DIA analysis Click Open The spot map s will automatically be added to the batch list DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Batch processor 6 E Removing To remove an item select the item from the BVA batch list select File Remove BVA batch item then confirm that the item is to be deleted Editing To amend the spot map attributes of an item in the BVA batch list select the desired item from the BVA batch list then select Edit Edit Item 6 4 Saving Data The batch list containing all the information entered into the Batch Processor can be saved by selec
56. list contains the relevant information to instruct the picker to excise the spots For more details refer to the relevant User Manual DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial IV Fully automated identification of differentially expressed proteins 13 Tutorial IV Fully automated identification of differentially expressed proteins 13 1 Objective This tutorial describes how to find statistically significant proteins that are differentially expressed between control and treated groups of bacterial cultures using DeCyder Differential Analysis Software in a fully automated manner The methodology of this tutorial can be applied to any two groups of protein mixtures with either replicate gels or replicate biological samples The tutorial outlines the various stages in experimental design sample organization protein detection and quantitation in a gel gel matching and statistical analysis 13 2 Overview The following list describes the various stages involved in identifying all the proteins that are differentially expressed in a given system and which may therefore be worthy of further investigation The stages are 1 An experimental design is devised that will generate statistically significant results a design which minimizes or eliminates in gel and gel to gel system variations 2 Eight sample lysates that form the basis of the experiment are prepared This consists of four lysates derived from f
57. makes the Linear pI calibration method most suitable Mw calibration is influenced by the second dimension gel used For example Log Linear Mw calibration method is most suitable for non gradient gels After choosing one of the three available options for calculating the pI and Mw click OK to perform the calculation The pI and Mw of the proteins should now appear in the Protein Table Displaying the Mw and pl values on the images To display the calculated pI and Mw values of the proteins on the gel images click on the Properties icon to display the Properties window By default the window should open on the Image View tab Select the Annotations check box then highlight the pl and Mw option from the list of different annotations Click OK to display these values on the images DeCyder BYA Properties Spot Map Table Match Table Protein Table Appearance Table Image View 3D View GraphView Database Colors Include in Image View IV Spots present in table only Spot Features IV Signature IV Annotation Master Number Protein ID Protein AC Protein Name Protein Comment and Mw Protein of Interest Filter Annotation IV Match vectors when Match Table is displayed Scrolling and Auto Center Settings IV Link image views when scrolling IV Auto center selected spots Picking References Primary Image View Default radius of picking references 5 100 30 Secondary Image View Default radius of picking references 5 100 30 Cancel
58. may be unacceptable for use in quantitative analysis and as a consequence DeCyder Differential Analysis Software only supports specific compression formats DeCyder Differential Analysis User Manual 18 1173 16 Edition AA B 3 A Tagged Image File Format TIFF images TIFF an extremely complex and flexible image format is used to exchange files between platforms and software applications The TIFF file consists of a number of labels tags that describe certain properties of the file such as gray levels color table byte format compression size etc After the initial tags comes the data which may be interrupted by more descriptive tags Although the TIFF file is an industry standard it has many variants DeCyder Differential Analysis Software has been validated for file formats generated by Amersham Biosciences imaging devices recommended for Etttan DIGE system applications The 16 bit TIFF format has 2 65536 levels of signal resolution and is the most commonly used file format for images The image below shows a schematic diagram of a TIFF file i Byte order s Version 8 byte structure located at 4 beginning of document Offset to 1 5 6 Image file Directory ee Directory Entry Public 32 bit offset 0 Tag 1 Tags 1 Image file Directory IFD Type 3 Each en x i ntry coun Tags ees Directory 2 Length 2 ytes Enyo 13 E Directory 14 mg Entry 1 25 Va
59. of the Image View Picking References The radius of the picking reference markers is entered in the dialog boxes 44 2 3 D View A three dimensional representation of the two images selected in the Image View localized on the selected spot This view is not visible in the Spot Map mode With the exception of the data displayed below each of the 3 D images the BVA and DIA 3 D Views are identical see section 3 4 2 A colored banner across one of the edges of the 3 D View images corresponds to the color of the title bar in the corresponding image view The banner reveals the orientation of the 3 D View image denoting the edge of the view closest to the top of the gel Pick locations when applicable are displayed in the 3 D View see section 5 6 Properties Properties associated with the 3 D View are defined in the 3 D View Properties window Selecting View Properties or selecting the Properties icon and then selecting the 3 D View tab displays the Image View Properties dialog box The size of area displayed in the 3 D View can be altered by entering a DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN positive integer between 3 and 80 The Show caption colors check box allows the colored banner indicating the upper edge of the 3 D View as seen in the Image View to be removed DeCyder BYA Properties Spot Map Table Match Table Protein Tab
60. of the internal standard and each sample within the same gel Because the internal standard is the same sample run within each gel this effectively normalises all the data These functions are performed within the Differential In gel analysis DIA module of the software B The Biological Variation BVA module is used to provide a quantitative com parison of protein abundance between all samples within the experiment DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Introduction to DeCyder Differential Analysis Software er 1 6 Steps involved in Image Analysis using DeCyder Differential Analysis Software Image analysis performed using DeCyder Differential Analysis software is performed using a number of complex algorithms some of which are patent pending They have been designed specifically for use with multiplexed 2 D images These steps can be broken into the following processes e Spot detection e Background subtraction e In gel normalization e Gel artifact removal e Gel to gel matching e Statistical analysis The complex algorithms associated with these steps form part of the in built functionality of the DeCyder Differential Analysis Software The various stages of gel processing are performed by different modules within the software suite 1 7 Structure of DeCyder Differential Analysis Software The software consists of four modules see Fig 1 4 DIA Differential In gel Analysis Protein spot
61. or two populations of groups e One Way ANOVA ANalysis Of VAriance Statistical analysis between all groups e Two Way ANOVA Statistical analysis between the two conditions in an experimental design where there are two independent factors e g time dose study This analysis allows the internal and mutual effects of the two factors to be quantified The log standardized abundance is the only variable subjected to the above statistical analyses within DeCyder Differential Analysis Software BVA The standardized abundance is derived from the normalized spot volume standardized against the intra gel standard The log of the standardized abundance values are used in order that the data points approach a normal distribution around zero thereby fulfilling the requirements of the subsequent statistical tests Consequently the statistical analysis functionality is not valid unless the experimental design includes an internal standard on every gel General requirements All spot maps included in the analysis need to be co run with an internal standard Only Spot Maps assigned as Analysis in the Spot Map Table are included in the statistical analysis Automatic recalculation Protein Statistics are recalculated automatically if any data that affects the statistics are changed e g if a match is broken or if a spot map is assigned to a new group DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 75 ED BVA Biologica
62. or type the path of the XML file containing the Detected Proteins in Gel LWS standard report into the Load XML file containing Detected Proteins in Gel LWS standard report field in the middle of the tool page 2 Select either View template spot map in separate window before saving or Save template spot map without viewing result 3 Click Create Ettan Protein Identification to start the conversion procedure At the end of the conversion procedure a Save As dialog is displayed Enter the name of the file where the Ettan protein identification file is to be saved Save the file in a location accessible by DeCyder Differential Analysis Software 150 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA LWS Integration 8 3 7 Importing protein data templates into the BVA module 1 Open the original BVA workspace and select File Import Protein Data Note Ifthe BVA module suspects that the file for importing originates from a different picking context than the current workspace a warning is displayed It is however still possible to import the information if desired but it is not recommended 8 8 Database queries in DeCyder BVA In DeCyder BVA it is possible to make online database queries based on the protein ID of the individual proteins The protein ID s generated by Ettan MALDI ToF Pro follow the notation used in the NCBI nr non redundant database To be able to use the database query function of DeCyder
63. protein abundance changes e High sensitivity and wide dynamic range 5 orders of magnitude e Minimization of system gel to gel variation e Easier matching between gels with increased confidence e Fewer gels required per experiment e Faster analysis due to fully automated gel processing workflow es DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 13 tg Introduction to DeCyder Differential Analysis Software 14 1 4 Experimental Design using an Internal Standard Ettan DIGE system provides the ability to multiplex samples enabling the use of an internal standard within each 2 D gel Ideally the internal standard should consist of a pool taken from all of the samples within the experiment The internal standard is labelled with one of the CyDye DIGE Fluor minimal dyes usually Cy2 if using CyDye DIGE Fluor minimal dyes or Cy3 if using CyDye DIGE Fluor saturation dyes and run on each gel in the experiment This creates an image that is the average of all experimental samples with all proteins in the experiment represented The presence of the internal standard in every gel provides an intrinsic link between samples Ettan DIGE system is currently the only 2 D gel electrophoresis protein difference analysis technique to utilize the internal standard approach There are several benefits of using an internal standard in 2 D experiments Firstly each protein spot in a sample can be compared to its representative within th
64. set parameters This step is optional Analyses can be performed without filtering workspaces to remove dust particles It is possible to omit filtering because a dust particle on a specific gel is very unlikely to be in the same position in other gels and therefore will not be matched and will not be in the final analysis However filtering does clean up the gel and may make the matching algorithm faster with possible greater accuracy DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 219 Tutorial IV Fully automated identification of differentially expressed proteins A 1341 Selecting gel images 1 220 Double click the DeCyder Differential Analysis Software DIA icon on your desktop to open the DIA module When the user interface appears select File Create Workspace ile i View Process Help Create Workspace Ctrl N Open Workspace Ctrl 0 Select Double Detection then click OK Type of Detection Single Detection one image Double Detection two images Triple Detection three images Cancel Browse to locate the folder Tutorial IV in the Tutorial data files folder Note All the images are named in the style Gel OX Cy X Standard Control or Treated Double click on image Gel 01 Cy2 Standard to load the Primary Gel Image Load Primary Gel Image Look in Tutorial Iv e E En My Recent E Gel 01 Cy3 Control Documents fe Gel 01 Cy5 Treated gel ti
65. showing the displacement of the spot should appear showing that the spots have been matched and the landmark has been set Note Landmarking can be aided by viewing the pattern around the selected spot in the 3 D View to confirm that the two selected spots do correspond to each other The number of spots being displayed around the selected spot in 3 D can be altered by opening the Properties window and selecting the 3 D View tab Altering the numerical value in the Spot Margin For Displayed Spot option adjusts the parimeter size which may consequently change the number of spots displayed around the selected spot It is recommended that landmarks be evenly distributed across the image as this aids the matching process Multiple gels can be landmarked in an identical manner Images from the same DIA file as a previously matched gel are automatically landmarked It is therefore only necessary to set landmarks in one image from a set of images derived from a single DIA analysis Ideally the landmarked image should be the standard spot map The landmarks which were set on previous spot maps will be colored yellow on the Master image thus the same landmarks can be set on the next match image by finding the spots which correspond to the yellow master image spots It is not necessary to set the same landmarks on each gel but as landmarks chosen by a user tend to be the clearest spots it is useful to have these highlighted so that the landmarking proces
66. spots on the image will still be detected even those outside the area of interest 1 Click on the Fit to window icon on the tool bar to fit the image to the Image View 2 Click on the Image view icon on the tool bar to have a full screen view of the gel image 3 Next click on the Properties icon to bring up the properties window DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 203 ED 1 Ill Processing the Preparative Gel and Generating a Pick List AA Select the Spot Display tab and deselect Similar Increased and Decreased so that the check boxes are identical to those shown in the figure below Click OK DeCyder DIA Properties 3D View TableView Colors Workspace Spot Display Image View Included spots FT Similar Decreased I Increased I Excluded spots IV Picking references and pick locations IT Picked spots 5 To set an area of interest select Edit Define area of interest Using the rectangular target pointer which now appears drag the mouse to draw a rectangle around the gel ensuring that edge artifacts are removed It does not matter if the Reference Markers are inside or outside the Area of Interest as these are set manually 204 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial Ill Processing the Preparative Gel and Generating a Pick List 12 6 Click on the Properties icon to bring up the properties window Select the Spot Display
67. tab reselect the Similar Increased and Decreased options and click OK 7 Select Process Exclude Filter and use the same parameters to filter the images again Exclude Filter All the spots on the outside of the area of interest will automatically be removed DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 205 ED 1 Ill Processing the Preparative Gel and Generating a Pick List AA 1234 Gel artifact removal A 3 D View clearly shows if a detected spot is a gel artifact rather than a protein spot due to the very steep sides and pointed top of an artifact compared to the smooth curve of a protein spot Actual Protein Dust Particle In order to exclude these artifacts from subsequent analysis a set of Exclude filter parameters must be generated 1 206 In the Table View click on the column header Area until the spot with the smallest area is present at the top of the table Max Slope Area Max Peak Max Volume Scroll down the table from one spot to another until the next spot in the table is an actual protein spot and not a dust particle or gel artifact Make a note of the Area value of the artifact immediately before the real protein spot This value will be used for the Area category in the Exclude filter Repeat this procedure for Volume When both values have been found select Process Exclude Filter to display the Exclude Filter dialog box Select the Area and Volume check boxes and ente
68. tests IV Average Ratio IV Student s T test Population 1 Population 2 Dption s Dption s Control Treated 4 Members 4 Members I One Way ANOVA between different groups r Cancel Help The spot specific statistics along with the average spot volume ratio now appear in the table DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial Il Employing an internal standard to Analyze Protein Changes Oy The statistical calculations are performed using the null hypothesis that the differences in protein standardized abundance i e the abundance relative to intra gel standard and or biological variation within groups of control and treated samples is not significant therefore a low value indicates a high level of significance 3 Click on the T test column header to order the table so that the lowest value is at the top of the table Click on the spot with the lowest value in the Table View This spot will now be displayed in all four views DeCyd BVA o o eated File Edit View Process Help Graph View Master No 1485 o Sa o ES a Standardised Log Abundance o 015 E E 3 i E i a a E Protein Table T test and Av Ratio Treated Control Pos Master No Status Protein ID Protein AC Appearance T test a I 2476 Unconfirmed 12 12 AM 8 9e 010 2 1426 Unconfimed 12 12 A M __2 8e 009 3 4 1331 Unconfirmed 12 12 AM 3 4e 0
69. the total amount of available memory including physical RAM is greater than 2 5 GB e Internet Explorer version 5 5 or higher must be installed to run the XML Toolbox module Note Avoid running other programs at the same time as the various DeCyder Differential Analysis Software modules 2 2 Installation DeCyder Differential Analysis Software is protected by a HASP key that should be attached to either the USB or parallel port on the computer when DeCyder Differential Analysis Software is in use The HASP device driver software is installed during installation of DeCyder Differential Analysis Software The color resolution must be set to at least 32 bit color and the screen resolution should be set to 1024 x 768 pixels These settings are accessed on the Settings tab in the Display icon on the Control Panel Local administrative privileges must first be obtained for installation of DeCyder Differential Analysis Software Insert the DeCyder Differential Analysis Software CD ROM and select the appropriate disc drive in Windows explorer From the files on the disc double click the Setup icon DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 23 ED Computer requirements and installation 2 2 1 Installation with a previous version present If a previous version of DeCyder Differential Analysis Software was installed select Reinstall all DeCyder components installed by the previous setup from the dialog box to remo
70. to analyze several gels in the DIA exclusively Alternatively multiple gels can be processed through both interfaces to produce a final BVA file XML Toolbox The XML Toolbox enables the extraction of user specific data from the XML files generated in either the DIA or BVA modules This data can be saved in either text or html format enabling users to access data in the DeCyder Differential Analysis Software workspaces for other applications The XML Toolbox also provides an interface for linking Ettan DIGE system data with Ettan Laboratory Workflow System 9 2 Scope of tutorials The following tutorials are aimed at introducing the functionality of DeCyder Differential Analysis Software within the context of an actual experiment The tutorials have been designed to be a step by step guide utilizing gel images and DeCyder Differential Analysis Software files which are co installed with the tutorials The four tutorials cover different aspects of the software suite They are all self contained and can be undertaken independently To assist the user each tutorial includes a completed version of the DeCyder Differential Analysis Software file which the tutorial is designed to generate These files all include the word finished in their names Tutorial I The DIA is used exclusively to demonstrate a small scale experiment to assess protein changes in samples that are limited See chapter 10 Tutorial II The second tutorial encompasses a sim
71. to extract specific data or exported to the BVA module for multi gel analyses Fig 1 5 Pick list for a single gel experiment o gt A XML file a EJ e Multi gel analyses using BVA DIA file Fig 1 5 Schematic representation of DIA module workflow Data extraction using XML Toolbox DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Introduction to DeCyder Differential Analysis Software er SaaS BVA module The BVA module utilizes the XML files exported from the DIA module together with the original gel images to match protein spots on different gels Data is then subjected to statistical analyses to accurately assess protein changes occurring between control and test groups within the experiment The results can be saved as BVA file which can be re opened in the BVA module Furthermore data can be exported in XML format for data extraction using the XML Toolbox Gel 1 1 XML file Pick list for a multi gel mage i GEL experiment TXT XML Gel 2 Image XMLfile GEL Gel 3 Image XMLfile GEL Data extraction using wa alee XML Toolbox BVA file Gel 4 Image XMLfile GEL Enea cea Fig 1 6 Schematic representation of BVA module workflow Batch Processor The Batch Processor integrates both the DIA and BVA modules enabling fully automated processing of multiple gels without user
72. to set landmarks on those images which are poorly matched The number of landmarks set depends on the accuracy of matching DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 189 ED 1 Il Employing an internal standard to Analyze Protein Changes 190 11 8 Statistical analysis 1 Click on the Protein Table icon or select View Protein Table The Protein Table lists each of the matched spots across all the images in the experiment The Protein Table mode consists of an Image 3 D Graph and Table View Each of the views are linked in such a way that selecting a spot in the Image View will display that spot in three dimensions highlight that spot in the table and graphically display the abundance values of that spot across all the images on which the spot occurs The Protein Table mode allows you to view those proteins that show a significant difference in expression between the control and the treated group The statistical level of significance is calculated using the T test if more than two groups the ANOVA test is used To perform statistical calculations select Process Protein statistics In the box entitled population 1 select Control in the box entitled population 2 select Treated Ensure that the Average Ratio and Student s T test boxes are selected as below Click Calculate Protein Statistics Type of statistical tests Independent tests normal Paired tests use sample ID Statistical
73. treated Experimental Group Paired test Abundance of a specific protein in the above five diseased individuals before and after drug treatment Data points are paired because the same individuals are present in pre and post treated groups Although there is no significant difference between the means of standardized abundance in the two groups a paired T test reveals that the two groups are significantly different since each individual exhibits an increase in protein abundance after drug treatment In this example there is a single result i e one image for each individual in each group Experiments employing replicate images from the five individual scan also be applied to a paired test In this instance the means of the replicas are calculated then subjected to pairing ensuring the pairs are independent Note The above graphs are conceptual not generated in DeCyder Differential Analysis Software In order to perform paired testing the pairing of spot maps must be pre assigned in the Spot Table view This can be done by inserting a numerical identifier in the text box labelled Sample ID at the bottom of the Spot Table view For example spot maps of protein samples from individual 2 in each the of groups can be assigned In this way the dependency can be associated with each spot map Group Sample ID Spot Map Comment Conte E 2 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 77 ED BVA Biologic
74. used as the starting center position of the model curve and all histogram data extending DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 251 252 from the tallest peak in both directions is included until the histogram value reaches below 10 of the main peak height At this point any data outside is excluded from the model curve fitting procedure This procedure is illustrated in the figure below The model curve parameters are then optimized to the selected histogram data using a standard Least Means Square gradient descent algorithm When the optimization is terminated the center of the model curve is denoted C Data used for normalisation 5 Normalize the primary spot map An assumption is made that the majority of the spots do not change hence the modal peak of the model histogram is normalized to occur at zero on the log volume ratio axis For this purpose the spot volumes for the primary gel are multiplied 10 C resulting in the mode volume for the primary and secondary being equivalent i e the spot volumes in the primary spot map are normalized using the following procedure Vas Vq 10 ii Where V4 is the resulting normalized volume of spot in the left or primary gel image The actual calculated volume of the detected spot is however never altered This modification of the volume is local to the normalization procedure and ensures that the model peak is shifted so that the majority of spots
75. variables determine a system s dynamic range pixel depth number of bits per pixel per color sensitivity of the image capture device i e CCD PMT accuracy of the focusing optics and precision of the measurement of the black and white points B 6 Image quality Visual image quality is the cumulative result of the scanning resolution the dynamic range of the scanned image and the scanning device or technique used Image quality is often expressed in terms of resolution but other factors also affect the quality of an image file Images are often stored at much higher quality than they are displayed on a monitor because most printing devices are capable of a much higher resolution than screen displays A key trade off in defining an appropriate level of image quality is the balancing of file size and resulting storage requirements with quality needs Since pixel dimensions and color depth of a graphic image are directly proportional to the file size of the image the higher the quality of an image the more storage space it will occupy High quality images also require more system resources e g higher bandwidth networks increased memory requirements and increased time and cost of the scanning process Effective image compression provides a key to maintaining quality while using less storage space and system resources However it is highly recommended that images be archived onto CD ROM to preserve storage space particularly if using a ne
76. with the gel DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 31 DIA Differential In gel Analysis Module 32 Double and triple detection algorithms are designed to take advantage of the inherent co migration benefits of the CyDye DIGE Fluor dyes A set of co run images two images in double detection and three images in triple detection is merged together thereby incorporating all spot features in a single image Spot detection and spot boundary definition is then performed using pixel data from all the individual raw images and the merged image The resultant spot map is overlaid back onto the original image files Since the spot boundaries are identical for all images the spots are effectively already matched This process results in highly accurate volume ratio calculations When creating workspaces the user selects the applicable algorithm and is subsequently prompted to load the appropriate number of image files Once the images are loaded the pre selected spot detection algorithm can be applied Select Process Process Gel Images to open the Process Gel Images window Process Gel Images Algorithm Selection Co Detection algorithm Version 5 00 01 Description Co detection algorithm Performs spot detection in one or more images based on features in the image contrast Estimated no of spots 2500 Autodetect Picking references D Cancel Help An estimation of the number of spots present o
77. 08 5 1195 Unconfirmed 12 12 A M 4 3e 008 6 1430 Unconfirmed 12 12 AM 4 5e 008 7 1459 Unconfirmed 12 12 AM 4 8e 008 8 1209 Unconfirmed 12 12 AM 6 0e 008 9 1135 Unconfirmed 12 12 AM 6 5e 008 10 1429 Unconfirmed 12 12 AM 7 1e 008 11085 Unconfirmed 12 12 AM 7 2e 008 12 1230 Unconfirmed 1202 AM 1 1e 007 13 1921 Unconfirmed 12012 AM 1 3e 007 14 2466 Unconfirmed 12 12 A M 1 3e 007 15 765 Unconfirmed 12 12 AM 1 9e 007 a Spot No 1298 Position 787 583 Spot No 1460 Position 673 565 s i j 8 Pri Spot Map Protein 1D Protein AC Name Comment T Pick I PTM il Y Confirm BES Mw Da IT Protein of Interest Ready Focus Primary 3D View NUM DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 191 ED 1 Il Employing an internal standard to Analyze Protein Changes 192 4 To alter the Graph View open the Properties window Click on the Graph View tab and ensure that the view parameters are identical to those shown below Click OK DeCyder BYA Properties Spot Map Table Match Table Protein Table Appearance Table Image View 3D View Graph View Database Colors Parameter Visualisation X axis Group zl Y axis Standardised Log Abundance Dashed Line Gel No Spot Color Group Include in Graph View IV Spots I Mean Value Crosses 7 Standards I Legend Y axis Settings Automatic C Manual Manual with automatic increase Max Min Cancel Ap
78. 1 Il Employing an internal standard to Analyze Protein Changes 11 5 Creating the BVA workspace 11 5 1 Selecting gels 1 Double click the DeCyder Differential Analysis Software BVA icon on your desktop to open the BVA workspace Select File Create workspace and browse to locate the XML files listed above in the folder Tutorial II located in the Tutorial data files folder Load DIA Result on XML format Look in Tutorial Il A mise ona Gel 01 xm My Recent Documents D My Network File name Gel 04 xml Gel 01 xml Gel 02 xml Gel 03 Places Files of type XML Result files xml y Cancel 2 Select all four XML files by holding down the shift key and clicking on each image using the left mouse button then click Open The XML and image files will automatically be loaded into the BVA workspace The BVA interface consists of four different modes Spot Map Table ST Match Table MT Protein Table PT and the Appearance Table AT Each screen in the BVA interface possesses a similar layout consisting of e Image View Displays the gel images making up the experimental set with the currently selected spot shown in magenta e Table View Depending on the screen selected displays either information on the images used in the experiment different properties for a single spot across the gels in the analysis set or protein spot specific statistical values e 3 D View Representation of t
79. 1 21 SpotNo 2309 Vi Unconfirmed gt 18 Table Vie Position 1143 1106P an ae H Y Pick pos A a gt T Pick T PIM Protein ID Comment Confirm F Protein of Interest I Exclude Focus Secondary 30 View NUM DeCyder Differential Analysis User Manual 18 1173 16 Edition AA DIA Differential In gel Analysis Module 3 2 Creating and opening workspaces Creating Workspaces DIA From the Desktop or Start button menu select the DeCyder Differential Analysis Software DIA icon to open the software module Create a new EL workspace by selecting File Create Workspace in order to load the images for analyzing Edit View Process Help Create Workspace Ctrl N Open Workspace Ctrl 0 There are three distinct algorithms employed for image spot detection which process different numbers of images simultaneously e Single detection one image e Double detection two images e Triple detection three images Select the detection algorithm appropriate for the number of images present on the gel being analyzed then click OK Type of Detection C Single Detection one image C Double Detection two images Triple Detection three images Cancel In the resulting window click on the image file that is to become the primary image and click Open Note DeCyder Differential Analysis Software has been validated for file formats generated by Amersham Biosciences imaging devices recommended for Ettan
80. 118 1371 1131 Unconfirmed 20 20 A M 0 019 1 15 1372 1132 Unconfirmed 20 20 AM 0 37 1 05 1373 1135 Unconfirmed 20 20 A M 0 00014 1 47 1374 1136 Unconfirmed 20 20 A M 6 5e 005 1 35 1375 1140 Unconfimed 20 20 AM 0 020 1 09 1376 1377 1143 Confirmed 20 20 AM 4 1e 006 1 47 1378 1146 Unconfimed 20 20 AM 0 00074 1 41 1379 1150 Unconfirmed 20 20 A M 0 047 1 14 1380 1155 Unconfimed 20 20 a M 0 0049 1 73 1381 1156 Unconfirmed 20 20 A M 0 37 1 03 1382 1158 Unconfirmed 20 20 A M 0 0095 1 09 1160 Unconfirmed 20 20 A M 0 96 1 00 y Fig D 2 Statistical data displayed for protein 1142 The independent and paired T test p values indicate that there is no significant difference in expression of protein 1142 from pre to post drug treatment groups The observed decrease in protein expression between experimental groups is not due to a consistent abundance change of this protein between individuals The independent and paired T test p values indicate that there is no significant difference in expression of protein 1142 from pre to post drug treatment groups p 0 066 and p 0 160 respectively The observed decrease in protein expression between experimental groups is not due to a consistent abundance change between individuals DeCyder Differential Analysis User Manual 18 1173 16 Edition AA D 2 Example of a two condition experiment Experimental objective An experiment is designed to investigate changes in protein ex
81. 144 ASKART wh Ai PAE A cir lee Sys Daas ea 142 OVERVIEW Picture sis pieis cette ii 141 Estimated Number of Spots cconocccccnccccococnncconooononcnnnonnnnnncnoninnns 32 Ettan DALT electrophoresis unit occccccccccnncnoninononanananannnnnnnos 32 Pitan DIGE System tna iaa iaa 12 Ettan Spot Handling Workstation ccccccccccccnnnninocononananannnnnnos 27 EttaiSpot Pleker Lusitania eta ali 27 Evaluation Tie captain dd 144 145 148 Bude Filter uti bind 44 203 Exporting data seta aia 49 50 106 Extract data tad o dh racic 130 F Frequency distribution rar ore itea satenii earned seg sd ene re Ven Ps 40 G Gel artifact removal cvs aid aii de 224 Generate PICK IST cacao ida areas orar 119 Generate standard report Detected proteins in gel 149 TAR A A Mat tle oe dds aka 71 Graphical usenintertace curia Bic a 51 GIQUPS cerda E a Ein 59 STS BIT ef SNEDE ERNE SOLER er BESES SEERE es hate ae ties 74 H SOS FAR A rn ts das aw dea bead aa 40 istograml VIEWS al in Dred 28 40 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA IMASEACGUISITION add id td Rs 243 Image files ud eo elk otis the 144 148 Image View ooooocccnoccncnoncnononcnononcnnnonononanancnnonncrananccnnnos 28 34 54 Immobiline DryStrip oconooccncccconoccnonononocanonocononononononannnnnononinnnos 32 Import data cadiz 114 Importing protein data templates into the BVA module 151 Independent analyses oo
82. 25 Matched Gel07 Standard Cy2 geCy2 2322 1243 A Standard 27 Matched Gel07 Standard Cy2 gecy2 2183 1270 A Standard 79 Matcher GAINA Standard v2 0 72 2NRA 1074 A Standard The experiment assesses two conditions strain and time consequently a Two Way ANOVA analysis is applicable The Two Way ANOVA check box must therefore be selected in the Protein Statistic dialog box to perform the statistical test Examples of the statistical outcome for selected spots are illustrated on the following pages CA DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 263 SSS Example proteins Graph Yiew Master No 545 o Q E a 3 5 v 2 Do pl a 3 E kel a Protein Table T test and Av Ratio 8 1 54 60 A M 0 011 46 60 A M P 0 00057 30 60 A M P 0 31 42 60 A M 0 83 40 60 A M P 0 46 44 60 A 0 47 52 60 A M 0 13 52 60 A M 0 60 4 60 A M 28 60 A M 0 47 48 60 A M P 0 40 48 60 A M 0 011 38 60 A M P 0 67 46 60 A M 0 12 12 60 A M Fig D 3 Statistical outcome for protein 545 The 2 ANOVA Strain and 2 ANOVA time values are not significant for protein 545 Therefore there is no strain to strain or duration of incubation effects on the expression of this protein 264 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Graph Yiew Master No 162 Protein ID Conalbumin Standardised Abundance Protein Table T test and Av Ratio 8 1
83. 500 No 5 04 10 P 100 V 1 Whole Image Pending Gel04Cy2StalCy2 Gel04Cy3 Tre Cy3 Gel 04 Cy5 Cor cy5 Min Gel4dia Gel4xml 2500 No 5 04 10 P 100 V 1C Whole Image Pending Pick gel Unas Min_ Pick d a Pick xml 2500 Yes 5 04 10 P 100 V 1 Whole Image Question Set BVA workspace Comment Ready 13 5 2 Assignment of spot map attributes To generate statistical data for the proteins that are expressed differentially between the control and treated groups it is necessary to identify the three different spot map groups control treated and standard within the software and to assign every image to one of the three groups The group names have to be created so that the appropriate images can be assigned to them 1 The Spot Map Assignment dialog box associated with the first spot map automatically appears after the previous step The standard spot maps will be automatically assigned to the standard group if the text Standard or Std is in the image name It is therefore always important to include the name of the group in the image name Click OK to confirm that the first spot map is an internal spot map 2 The Spot Map Assignments dialog box for the second spot map then appears The second spot map will be derived from either a control or treate
84. 60 A M P 0 0061 1 12 49 341 Unconfirmed 58 60 A M 0 0062 1 07 50 1035 Unconfirmed 36 60 A M 0 0062 2 94 Unconfirmed 54 60 A M P 0 0066 1 13 A Dosente od andan nana car se Specific Requirements If there are at least two members in each group T tests and average ratios will be calculated If there is only one member in each group only the average ratio will be calculated 4 8 6 ANOVA Overview Analysis of variance ANOVA is a family of methods that can be used to analyze the results from both simple and complex experiments It is one of the most important statistical tests available for biologists and at its lowest level it is essentially an extension of the logic of Student s T tests to those situations where the concurrent comparison of the means of three or more samples is required Thus when comparing two means the ANOVA will give the same results as the T test for independent samples if comparing two different groups or observations There is no restriction on the number of groups that can be analyzed It is equally valid for testing differences between 2 groups or among 20 There are two types of ANOVA analyses in DeCyder Differential Analysis Software BVA module One Way and Two Way The One Way ANOVA evaluates differences in all assigned groups whereas the Two Way ANOVA can also evaluate the statistical significance of effects DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 81
85. 8 1173 16 Edition AA 147 ED LWS Integration 148 8 5 3 After XML conversion In Ettan LWS the converted pick list file from section 8 5 2 will now be used as the evaluation file together with the gel image file in sample definition for the 2D gel see section 8 4 Note If you have entered the sample generated within Ettan DIGE system as a tube see section 8 3 follow the normal workflow in Ettan LWS using the converted pick list file as the evaluation file in Image Analysis on the Image Results tab After entering the pick lists in Ettan LWS do not modify the original DeCyder BVA workspace If changes are made to the BVA workspace between exporting the pick list data and importing the annotated Ettan protein identification result it cannot be guaranteed that the imported protein IDs are still consistent with the information in the BVA workspace If changes are required it is advisable to make these changes on a copy of the original 8 6 XML Toolbox Protein Identification The Protein Identification tool is used to transform Ettan LWS data back into DeCyder Differential Analysis Software format It is then possible to compare the original gel images in DeCyder BVA with the protein identification results from the MALDI mass spectrometry analysis in Ettan LWS The report Detected proteins in gel from Scierra LWS saved in xml format is used as the input file for the Protein Identification tool This file is converted to
86. A by selecting File Export Workspace or in DIA by selecting File Export Spot Map The file must be saved to a location accessible by the XML Toolbox DeCyder Differential Analysis User Manual 18 1173 16 Edition AA LWS Integration 8 E 852 Using the LWS Pick List tool A DeCyder XML Tools Microsoft Internet Explorer File Edit View Favorites Tools Help A Q rex x Ej a j Search fg Favorites media s C Program Files Amersham Biosciences DeCyder XMLToolbox htm DeCyder LWS Pick List XML Toolbox Load XML file generated from DIA or BVA GA2D DIGE EttanProteinidentification files Proteinidentifice Tab Separated Tables File type BVA Web Tables OE LWS Pick List View pick list in separate window after saving s O save pick list without viewing result Protein Identification Create Pick List Help 3 My Computer 1 Either browse for a file or type the path of the file into the Load XML file generated from DIA or BVA field at the top of the tool page 2 Select either View pick list in separate window before saving or Save pick list without viewing result 3 Click Create Pick List to start the conversion procedure At the end of the conversion procedure a Save As dialog is displayed Enter the name of the file where the pick list is saved Type the extension xml to the file name Save the file in a location accessible by Scierra LWS DeCyder Differential Analysis User Manual 1
87. Analysis User Manual 18 1173 16 Edition AA Batch processor 6 Click Yes to set the BVA workspace in order to assign spot map attributes and set up statistical analysis and generate a pick list if required 6 2 2 Setting up the BVA Batch List The Spot Map Assignment dialog box appears when setting up the BVA workspace Item 1 Gel01 Standard Cy2 gel Spot Map Assignments Spot Map function IT Master IT Template IT Pick Experimental Design Group Standard Group description Condition 1 Condition 2 Sample ID Spot Map Comment to As with the Spot Map Table mode in the BVA module attributes i e spot map function group conditions sample ID and spot map comments can be assigned to individual spot maps Attribute assignment is performed in the above dialog box Attributes are assigned sequentially to each spot map by selecting the appropriate spot map function or entering the desired experimental design criteria Click OK once all the attributes are assigned to the first spot map The Spot Map Assignment dialog box for the second spot map will automatically appear The spot map attributes for all spot maps can therefore be assigned in this iterative process All attributes are assigned as in the Spot Map Table mode section 4 5 1 and 4 5 2 In the Batch Processor groups are added by selecting the Add button in the Spot Map Assignment dialog box Note As with the BVA module if no master
88. BVA based on the protein ID s from Ettan MALDI ToF Pro a new query definition that matches the NCBI nr format must be added in the database property page When adding a new database query definition enter information in the two fields in the dialog Edit web database label and address 1 In the Label field type NCBI nr Protein search based on ID or something similar 2 In the Address field type the following search string http www ncbi nlm nih gov entrez eutils efetch fcgi rettype gp amp retmode html amp db protein amp id BVA_PROTID 3 Click OK DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 151 ED LWS Integration 152 Add new web database label and address Label NCBI nr Protein Search based on ID Address A placeholder string has to be included in the address and will be replaced by the protein s ID or AC Eg http www expasy ch cgi bin get sprot entry BV4_ PROTID BVA_PROTIDH Protein ID HBVA_PROTACH Protein AC BVA_PROTIDACH Both Protein ID and AC gov entrez eutils efetch fegi rettype gpkretmode html amp db proteinkid HBVA_PROTID Test Select a Protein ID or AC that will Test replace the placeholder string zl Cancel Help Fig 8 7 Information to enter in the dialog Edit web database label and address when adding the new database query definition For further information on using the database query function and adding new database query definitions s
89. Cy3 Treated gelt DIGE Min Cy3 2298 1081 A Treated BA treated 3 Matched Gel 04 Cy5 Control gel 1 DIGE Min CyS 2298 1081 A Control Untreated 4 Matched Gel 01 Cy2 Standard g2 DIGE Min Cy2 2254 1130 A Standard 5 Matched Gel 01 Cy3 Control gel2 DIGE Min Cy3 2254 1130 A Control Untreated 6 Matched Gel 01 Cy5 Treated ge2 DIGE Min Cy5 2254 1130 A Treated BA treated 7 Master Gel02 Cy2 Standard g3 DIGE Min Cy2 2572 2572 MA Standard 8 Matched Gel 02 Cy3 Treated gel3 DIGE Min Cy3 2572 2572 A Treated BA treated 9 Matched Gel 02 Cy5 Control gel 3 DIGE Min CyS 2572 2572 A Control Untreated 10 Matched Gel 03 Cy2 Standard g DIGE Min A Standard 11 Matched Gel 03 Cy3 Control gel4 DIGE Min A Control Untreated Note A pair of images from the same gel will have the same number of spots since the DIA detection algorithm is designed to detect the same number of spots on image pairs from the same gel DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 209 ED 1 Ill Processing the Preparative Gel and Generating a Pick List Under the column Function all the images will be labelled with the function Analysis A and one of the analysis images the image with the largest number of detected spots will be labelled with the function Master M The analysis function designation indicates that each of the images will be included in the post matching statistical analysis 6 The Master function can be assigned to a different image by selecting the
90. Cyder Differential Analysis User Manual 18 1173 16 Edition AA 185 ED 1 II Employing an internal standard to Analyze Protein Changes 186 been set on the first image select the second spot map from the Select Match Gel scroll down menu The landmarks which were set on the previous match image will be colored yellow on the Master image thus the same landmarks can be set on the next match standard spot map by finding the spots which correspond to the yellow master image spots Note Landmarks are only required on the standard image from a set of images that have been simultaneously processed in a DIA analysis Repeat this procedure until landmarks have been set on all the standard images from each DIA analysis When landmarking is completed click on the Landmark mode button to exit the landmark mode Click on the Match icon ensure that Match all is selected and click Match to commence the matching process Note It is usually only necessary to set landmarks on those images that differ significantly from the Master image In such cases it may be necessary to set more landmarks In addition landmarks may have to be set after matching if the number of matched spots is low or if there are a high number of wrongly matched spots in which case matching will have to be repeated 6 Assessing matching Double click on the Match Table icon or select View Match Table This screen displays data on the gel to gel matching of the differe
91. Differential Analysis User Manual 18 1173 16 Edition AA 67 ED BVA Biological Variation Analysis Module 68 Furthermore each co detected spot for multiple images on the same gel all possess the same match quality value which is associated with the profile of the internal standard spot only 4 6 5 Spot Merging Very occasionally a protein peak will be dissected into several spots despite appearing as a single discrete spot This is normally due to subtle gradient changes on the spot surface that are not always apparent to the naked eye These split spots can be merged to form a single spot boundary within the Match Table mode Select the matched spot in the image view that requires to be merged to surrounding spot s Selecting Edit Merge Spots results in the surrounding unmatched spots being exclusively displayed Click on the spot in the image view that requires merging to the spot selected originally Note Two spots can be merged if at most one of them is matched to another spot If one master spot is matched to several spots it s possible to merge it with another spot but only if that other spot is not matched DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN Both the image view and the 3 D View display the new spot boundary showing all the component spots The volume of the merged spot a
92. GE Fluor dye types is identical Here in tutorial II a preparative gel for a CyDye DIGE Fluor minimal dye experiment is used to demonstrate the various steps in generating a pick list The aim of this experiment is to generate a pick list on a SYPRO stained gel relating to the most significant differentially expressed proteins in control and treated bacterial lysates determined from Ettan DIGE system analytical gels When using the CyDye DIGE Fluor minimal dyes a SYPRO stained pick gel is required rather than picking directly from an Ettan DIGE system gel because the CyDye DIGE Fluor minimal dye label only 5 of the protein This results in the majority of the unlabelled protein migrating slightly different and hence not ending at exactly the same place as the labelled protein seen on the gel Therefore a post stain total stain is used on a preparative gel for spot excision The image generated is then matched to the analytical set The positions of the significant differences are transferred to the SYPRO stained gel and a pick list is created This pick list along with the SYPRO stained gel is transferred to the automated Ettan Spot Picker or Ettan Spot Handling Workstation for spot excision and subsequent digestion spotting and mass spectrometry analysis DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 197 ED 1 Ill Processing the Preparative Gel and Generating a Pick List 1 12 2 Overview This Tutorial fo
93. Gel CyDye DIGE Fluor Strain Time Group minimal dye Condition 1 Condition 2 1 1 Cy3 I I I 2 2 Cy3 I I I 3 3 Cy3 I I I 4 1 Cy5 T 2 2 5 2 Cy5 I 2 2 6 3 Cy5 I 2 2 7 4 Cy3 I 3 3 8 5 Cy3 I 3 3 9 6 Cy3 I 3 3 10 4 Cy5 1 4 4 11 5 Cy5 1 4 4 12 6 Cy5 1 4 4 13 7 Cy3 2 1 5 14 8 Cy3 2 1 5 15 9 Cy3 2 1 5 16 7 Cy5 2 2 6 17 8 Cy5 2 2 6 18 9 Cy5 2 2 6 19 10 Cy3 2 3 7 20 11 Cy3 2 3 7 21 12 Cy3 2 3 7 22 10 Cy5 2 4 8 23 11 Cy5 2 4 8 24 12 Cy5 2 4 8 The above parameters can be designated either in the DeCyder Differential Analysis Software Batch Processor or from DeCyder Differential Analysis Software BVA The resultant Spot Map Table is illustrated on the following page S 262 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Spot Map Table Gel03 Cy3 gel Cy3 2359 932 10 Matched A i Strain 1 0 mins 1 1 4 Matched Gel01 CyS gel Cy5 2182 943 A 2 Strain 1 30 mins 1 2 8 Matched Gel02 Cy5 gel cys 2097 871 A Strain 1 30 mins 1 2 12 Matched Gel03 Cy5 gel cys 2318 992 A 2 Strain 1 30 mins 1 2 14 Matched Gel04 Cy3 gel Cy3 2219 1281 A 3 Strain 1 60 mins 3 18 Matched Gel0S Cy3 gel Cy3 2085 2085 A 3 Strain 1 60 mins 1 3 22 Matched Gel06 Cy3 gel Cy3 2244 1260 A 3 Strain 1 60 mins 1 3 16 Matched Gel04 Cy5 gel Cy5 2288 1284 A 4 Strain 1 90 mins 1 4 20 Matched Gelos CyS gel Cy5 2256 1795 A 4 Strain 1 90 mins 1 4 24 Matched Gel06 Cy5 gel cys 2303 1295 A 4 Strain 1 90 mins 1 4 26 Matched
94. Gel07 Cy3 gel Cy3 2322 1243 A 5 Strain 2 O mins 2 1 30 Matched Gel08 Cy3 gel Cy3 2086 1074 A 5 Strain 2 O mins 2 1 34 Matched Gel09 Cy3 gel Cy3 2061 1097 A 5 Strain 2 O mins 2 1 28 Matched Gel07 Cy5 gel Cy5 2183 1270 A 6 Strain 2 30 mins 2 2 32 Matched Gelos Cy5 gel Cy5 2418 1289 A 6 Strain 2 30 mins 2 2 36 Matched Gel09 Cy5 gel cys 2349 1183 A 6 Strain 2 30 mins 2 2 38 Matched Gell0 Cy3 gel Cy3 2272 1155 A 7 Strain 2 60 mins 2 3 42 Matched Gel11 Cy3 gel Cy3 2255 936 A 7 Strain 2 60 mins 2 3 46 Matched Gel12 Cy3 gel Cy3 2111 1116 A 7 Strain 2 60 mins 2 3 40 Matched Gel10 Cy5 gel cys 2142 1208 A 8 Strain 2 90 mins 2 4 44 Matched Gel11 Cy5 gel cys 2176 892 A 8 Strain 2 90 mins 2 4 48 Matched Gel12 Cy5 gel Cy5 2459 1214 A 8 Strain 2 90 mins 2 4 1 Matched Gel01 Standard Cy2 geCy2 2304 956 A Standard 3 Matched Gel01 Standard Cy2 gecy2 2182 943 A Standard 5 Matched Gel02 Standard Cy2 geCy2 2163 891 A Standard 7 Matched Gel02 Standard Cy2 geCy2 2097 871 A Standard 9 Matched Gel03 Standard Cy2 gecy2 2359 932 A Standard 11 Matched Gel03 Standard Cy2 gecy2 2318 992 A Standard I 13 Matched Gel04 Standard Cy2 geCy2 2219 1281 A Standard 15 Matched Gel04 Standard Cy2 gecy2 2288 1284 A Standard 17 Master Gel05 Standard Cy2 geCy2 2085 2085 MA Standard 19 Matched Gel05 Standard Cy2 geCy2 2256 1795 A Standard 21 Matched Gel06 Standard Cy2 gecy2 2244 1260 A Standard 23 Matched Gel06 Standard Cy2 gecy2 2303 1295 A Standard
95. In order to exclude these artifacts from subsequent analysis a set of Exclude filter parameters must be generated 1 Inthe Table View click on the column header Max slope until the spot with the highest max slope is present at the top of the table Max Slope Area Max Peak Max Volume 166 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial Using DIA module for preliminary investigation of protein changes 10 E Scroll down the table from one spot to another until the next spot in the table is an actual protein spot and not a dust particle or gel artifact Make a note of the Max slope value of the artifact immediately before the real protein spot This value will be used for the Slope category in the Exclude filter 3 Repeat this procedure for Area Max Peak Height and Max Volume but in each case ordering the table so that the smallest value is at the top of the table 4 When all the values have been found select Process Exclude Filter to display the Exclude Filter dialog and enter the found values Click OK Exclude Filter Spot properties FT Filter confirmed spots IV Slope gt 1 1 IV Area lt 100 IV Peak Height lt 1100 IV Volume lt 10000 Current Area of Interest x direction 8 2096 Y Position 16 1806 Cancel Help An example of a non stringent set of filter parameters that can be used to run a light filter is as follows Slope 1 1 Area 100 Peak Heigh
96. Integration of DeCyder Differential Analysis Software with Ettan DIGE system experimental design DeCyder Differential Analysis Software was developed specifically for the 2 D DIGE methodology and therefore all the advantages of this approach are utilized in the software e DeCyder Differential Analysis Software in conjunction with Ettan DIGE system allows the analysis of experimental designs with various degrees of complexity A simple control treated experiment through to a multi condition experiment addressing factors such as dose and time can be performed in a single analysis e The relationship between any number of samples can be accurately quantified and statistically analyzed in DeCyder Differential Analysis Software by employing the internal standard see Fig 1 2 This approach results in unparalleled accuracy allowing experimental conclusions to be drawn with high confidence No other 2 D electrophoresis technique available is capable of resolving multiple samples in this manner and hence Ettan DIGE system is unique in exploiting use of an internal standard on every gel e The novel co detection algorithm exploits the identical spot patterns generated when multiple samples are resolved on the same gel The algorithm generates identical spot detection patterns on all images derived from the same gel Hence all spots on the same gel are effectively matched with the identical spot boundaries e Spot quantitation is performed aut
97. Interest 1 only IT Proteins assigned as Pick only Table Column Order and Visibility Select columns to display and the column order by drag and drop Left Column MP Master No Status Protein ID Protein AC MaAppearance Paired T test Paired Av Ratio M1 RMANOVA M2 RMANOVA Condition1 V 2 AMANOVA Condition2 Vi2 AMANOVA Interact VPicked W Pick Spot Vol Function Comment Match Quality Right Column WIPTM Restore to default order and selection Default Cancel Apply Help The Function column header in the Protein Table then displays the proteins with protein of interest status possessing I status in the function column The POI assigned proteins can then be reviewed sequentially to ensure that they are correctly matched and suitable for picking Once the spot is deemed suitable for picking select the Pick check box in the data control panel Once the spot is assigned with a pick status it will have a black spot boundary in the image view and the letter P in the Picked column of the Protein Table The pick location will then need to be reviewed on the spot as described below before reviewing the next spot in the table DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 115 ER Spot picking 116 5 6 Editing Pick Locations In the BVA module proteins assigned with pick status can be shown exclusively in the image view when i
98. M 0 0025 0 52 0 43 al AL 723 22 60 A M P 445 722 54 60 A M 0 85 0 29 0 55 446 447 720 46 60 A M 0 38 0 67 0 20 445 719 50 60 A M 0 42 0 21 0 57 449 718 44 60 A M 0 78 0 039 0 10 450 717 36 60 A M P 0 68 0 41 451 716 52 60 A M 0 25 0 40 0 51 715 38 60 A M 0 78 0 11 0 79 58 60 A M P 0 45 0 73 Fig D 5 Statistical outcome for protein 721 The 2 ANOVASStrain and 2 ANOVA time values are statistically significant for protein 721 Therefore there are strain specific changes in expression of this protein The expression of this protein also changes significantly in at least one timepoint Moreover there is a significant interaction 2 ANOVA Interaction lt 0 01 between strain and time whereby there is a decrease in expression of protein 721 over time that is specific to one strain DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Appendix E DeCyder Differential Analysis Software keyboard shortcuts E 1 DIA module keyboard shortcuts Spot Controls Alt C Confirm spot Alt P Previous Alt N Next Alt Include Alt X Exclude Alt K Pick Alt S Spot ID Alt M Comment Workspace Alt F Open File menu Alt E Open Edit menu Alt V Open View menu Alt R Open Process menu Alt H Open Help menu File menu Ctrl N Create new workspace Ctrl O Open workspace Ctrl S Save
99. NOVA testing Performing analysis 1 88 In the Protein Statistics dialog box select the option button to indicate whether the test is independent or paired Pairing of spot maps has to be pre assigned in the Spot Map Table View see section 4 8 4 for paired testing Select the Two Way ANOVA check box Click Calculate Select the Properties icon on the tool bar and ensure that 2 ANOVA condition 1 2 ANOVA condition 2 and 2 ANOVA interact check boxes are selected in the spot map tab If the Two Way ANOVA analysis is paired select the Paired tests option button A repeated measures paired Two way ANOVA will then be automatically calculated Click Calculate to perform the analysis DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN Protein Statistics Type of statistical tests Independent tests normal Paired tests use sample ID Statistical tests I Average Ratio F Student s T test Population 1 Population 2 Dption s Dption s 3 Members 3 Members I One Way ANOVA between different groups IV Two Way ANOVA between Dose and Time Calculate Cancel Help The Two Way ANOVA p values will then be displayed in the protein table As with the other analyses spots can be ordered by significance by clicking on the appropriate 2 ANOVA column headings in the protein table Specific requirements At least two experimental groups with
100. Processing the Preparative Gel and Generating a Pick List 212 The position of the target can be altered by selecting Edit Edit picking Reference Place the hand shaped cursor that now appears on the target and hold down the left mouse click Move the target so that it fits exactly around the reference Magnifying the area of the gel around the reference marker by selecting the Zoom in icon may make accurate alignment easier After reviewing the left reference marker zoom into the right reference marker then check and edit its position in an identical manner Select Edit Edit picking References to exit this mode mel A When the picking references have been reviewed it is necessary to match the preparative gel to the master image thereby matching the preparative image to all the analytical images 12 4 3 Matching and landmarking 1 To match the images first move to the Match Table by clicking on the Match Table icon From the images it is clear that the SYPRO image is slightly different from the analytical images It is therefore necessary to set manual matches known as landmarks Landmarking allows the user to manually define matched protein spots in order to improve the accuracy of the gel to gel matching process Click on the Magnify Image View icon to display the gel images only The image on the left represents the Master image while the image on the right represents one of the images to be matched to the master Ens
101. Properties icon and select the Spot Display tab and reselect the Increased Decreased and Similar options and click OK Note To remove the area of interest select Edit Remove area of interest Creating an Exclude Filter The Exclude Filter removes specific spots based on their physical characteristics in order to eliminate non proteinaceous spots from further analyses Select Process Exclude Filter or select the icon from the toolbar to open the Exclude Filter dialog box Exclude Filter Spot properties FT Filter confirmed spots IV Slope gt pio IV Area lt ho M Peak Height lt fioo IV Volume lt fioo Current Area of Interest Whole Image Cancel Help The 3 D View generally reveals if a detected spot is a gel artifact rather than a protein spot Dust particles normally have very steep sides and a pointed peak when compared to the smoother curve of a protein spot Therefore dust particles have high slope values and low area values DeCyder Differential Analysis User Manual 18 1173 16 Edition AA DIA Differential In gel Analysis Module E The figure below illustrates a protein spot and a dust particle spot Actual Protein Dust Particle Irregularities in the gel surface may also be detected as a spot however such artifacts have low peak height and volume values Artifact spots can therefore be excluded on the basis of slope area peak height and volume The appropriate filter parameter values can be
102. Similar Unconfirmed 2316 Similar Unconfirmed 2315 Decreased Unconfirmed Similar Unconfirmed Decreased Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Decreased Unconfirmed Similar 1 92 Unconfirmed Similar 1 21 0 Volume 38568005 SpotNo 2309 Y Unconfirmed Similar Table Vie t882 Position 1143 11061 Similar 18 v Pi Tets Area 1419 Pick pos Ar ig gt T Pick T PIM Protein ID Comment Confirm I Exclude IT Protein of Interest Focus Secondary 3D View NUM The user interface is divided into four main windows which show the Image View the Histogram View the 3 D View and the Table View 222 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial IV Fully automated identification of differentially expressed proteins The four views are linked Clicking on any spot on the Image View highlights the spot in magenta in the Histogram View the Histogram View is disabled when analyzing single images such as SYPRO stained gel images The spot is also represented three dimensionally and is highlighted in gray in the Table View Each of the four views can be expanded to fill the whole screen To do this click on the View button and the menu below is displayed View Process Help Area in 3D Ctrl A Rotate 3D Ctrl R Zoom gt Contrast Brightness Alt B Image View Histogram View 3D View Table View A
103. Suma proteomics DeCyder Differential Analysis Software Version 5 0 User Manual Amersham am 18 1173 16 AA e Y Biosciences Terms and Conditions of Sale Unless otherwise agreed all goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group which supplies them A copy of these terms and conditions is available on request Trademarks Amersham and Amersham Biosciences are trademarks of Amersham plc Cy CyDye DeCyder Drop design Ettan Immobiline IPGphor PlusOne Scierra and Typhoon are trademarks of Amersham Biosciences Limited HASP is a trademark of Aladdin Knowledge Systems Ltd Microsoft Windows Word Excel Internet Explorer are trademarks of Microsoft Corporation OpenGL is a trademark of Silicon Graphics Inc Pentium is a trademark of Intel Corporation or its subsidiaries Spotfire is a trademark of Spotfire Inc SYPRO is a trademark of Molecular Probes Inc Patents and Licences CyDye 2 D Fluorescence Difference Gel Electrophoresis 2 D DIGE technology is covered by US patent numbers US6 043 025 US6 048 982 US6 127 134 and US6 426 190 and foreign equivalents and exclusively licensed from Carnegie Mellon University CyDye this product or portions thereof is manufactured under licence from Carnegie Mellon University under US patent number US5 268 486 and other patents pending The purchase of CyDye fluors includes a
104. TTUCIULO a dia 52 4 25 Protein quantitation sasni tiaia a AAN 53 4 3 Creating and Opening workspaces ococooccccnnnnnncnnnnninonininininininnnos 53 4 3 1 Creating WOrkSPACES ocoooocononononoricincananananananananan aran nononenons 53 4 3 2 Opening workspaces ooocooocnnonononoconononononnnnananananananononnononnnnos 54 AA VIEWING SOOE Calta 10d dis 54 4 4 1 MALO VICW Scala iii ii 54 A SED VOW ensenen e a a e a 56 4 4 3 Table VW ti da 57 4 5 Defining spot map attributes occcnnnicocucoooooooooonnnnnnnnnnannnnnnnnninnnns 58 4 5 1 FPUACTION ASSIGNINGN Lili a td ibas 58 4 5 2 Group assignment cccccccnccanncccnnnonnnnononononnnonoconnononnncnnoncnnnnnnnos 59 4 5 3 Commentassig AMenE fasan eiar asah dl bat 61 4 5 4 Spot Map Table Mode ooccccccoooocccccoconannccnononanancnornnananos 61 4 5 5 Spot Map Table properties cccoooooncccccononacoononanananononcnans 62 4 6 Inter gel Matching cccccccnnnnnanonononenananannnnnnnnonnnnnnnnoninnnninnannnns 63 4 6 1 PANGINGCKING st sd alce lat Loa 63 4 6 2 MATICES A A A AA 65 4 6 3 WENA EA O 65 4 6 4 Match Quality MetriC isaimini sia 67 4 6 5 POEMA al oie 68 4 6 6 Match Table dia dieta 70 4 6 7 Match Table properties ccccmooononocononananonanannonononnnnnnnoos 70 4 7 Graphical representation ccccnnnnnnnnnononoconanannnnnnnnonononononononnnnns 71 4 8 PROLEIMSLALISHICS A oie 73 4 8 1 Opening the protein statistics dialog BOX esse 73 4 8 2 DEMAS BIOU PSH t
105. Table select the spot in the Table View The spot is automatically selected in all the other views In the Secondary image scroll down menu above the image select the image that corresponds to the Master image Zoom into the area of the spot in the Image View as described previously to determine whether the correct spots have been matched correctly between the primary and the secondary images If this is the case select another image using the Primary image scroll down menu and repeat the match assessment 2 Ifthe protein spots are incorrectly matched move to the Match Table View by clicking MT and alter the match as described previously 3 When all the matches are correct the protein spot can be confirmed by clicking the Confirm button This records that the spot has been manually checked This confirmation should be done for all the significant differences that have been assigned with a protein of interest status If after manual scrutiny the user decides that the spot should not be selected deselect the protein of interest status for the given spot by checking the box marked Protein of Interest at the bottom of the Protein Table 11 9 3 Exporting the Pick List and physically excising spots from the gel To excise the spots from the gel a separate preparative gel should be used This is matched to the analytical gel set and the pick status is eventually transferred to the relevant matching spots on the preparative gel The pick list with
106. ToF Pro 240 V 18 1156 53 2 D Electrophoresis Principles and 80 6484 89 Methods Ettan DIGE Training CD 18 1164 39 Typhoon Variable Mode Imager For product information please inquire with your local Amersham Biosciences sales office Table continued on next page DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Appendix F Related products and documentation 271 272 Item Product Code CyDye DIGE Fluor Cy2 minimal dye RPK 0272 25 nmol CyDye DIGE Fluor Cy3 minimal dye RPK 0273 25 nmol CyDye DIGE Fluor Cy5 minimal dye RPK 0275 25 nmol CyDye DIGE Fluor Cy2 minimal dye 25 8008 60 10 nmol CyDye DIGE Fluor Cy3 minimal dye 25 8008 61 10 nmol CyDye DIGE Fluor Cy5 minimal dye 25 8008 62 10 nmol CyDye DIGE Fluor Labelling Kit for Scarce 25 8009 83 Samples CyDye DIGE Fluor Labelling Kit for Scarce 25 8009 84 Samples plus Preparative Gel Labelling PlusOne Urea 17 1319 01 PlusOne CHAPS 17 1314 01 PlusOne DTT 5 g 17 1318 02 Ettan 2 D Quant kit 80 6483 56 Ettan 2 D Clean Up kit 80 6484 51 PlusOne Bromophenol Blue 17 1329 01 PlusOne Tris 17 1321 01 PlusOne SDS powder 17 1313 01 PlusOne Glycerol 87 17 1325 01 PlusOne ReadySol IEF 40 T 3 C 17 1310 01 PlusOne TEMED 17 1312 01 PlusOne Ammonium Persulphate 17 1311 01 PlusOne Glycine 17 1323 01 Ultrapure DMF US14862 DeStreak Rehydration Solution
107. Way ANOVA exam pl aru ng a a aen lee eu 261 U serman al id A dd 11 User defined protein labelling coonmoncooninnonoroononrnnorennnrrnnnnenos 94 V A a da ead av ae ei ts 33 WwW Workspace COLO E E E E E 29 OPEN errata dla nba 30 AA DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 283 Properties casta an 31 X AML a A ii 129 Tas definitions usan Aita 132 XML Tool b k ar aid 129 Z TANTA DEEE ASE A S EEES RE Ee EAA teens EO ET EAA AT 34 yA 284 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Amersham e Biosciences TC information Uppsala Printed in Sweden by TK tryck AB Uppsala
108. a user defined delay can be introduced however the batch processor must be left open during the delay period Note The Master can also be set when assigning the spot map attributes by selecting the Master check box for the desired master spot map The following screen is displayed while the Batch Processor is working El DeCyder Batch Processor DIA Di C Fiman Tutorial images v5 Temp Tutoriald Browse BVA Filename CAFimankTutorial images vo Temp Tutorial Set XML Dic C MFiman Tutorial images v5 Temp Tutoriald __ Browse Pick Filename C Fiman Tutorial images v Temp Tutorial Set Status Primary Label Secondary Label Tertiary Label Type DIA File XML File JEstimated Pick ref Found Exclude Fiter Area of interest Gel 01 Cy2StalCy2 Gel 01 Cy3 Cor Cy3_ Gel01 Cy5 Tre Cy5 Min Geltdia Gel 1 xml 2500 2254 5 0 A 100 P 100 V 1C Whole Image Gel 02 Cy2 Stal Cy2 GelO2Cy3Tre Cy3 Gel 02 Cy Cor Cy5 Min Gel2dia Gel 2xml 2500 2572 5 0 4 100 P 100V 1C Whole Image Gel 03 Cy2 Stal Cy2 Gel 03 Cy3 Cor cy3 GelO3Cy5Tre Cy5 Min Gel3dia Gel3 xmi 2500 0 4 100 P 100 V 1 Whole Image Gel 04 Cy2 Stal Cy2 Gel04Cy3Tre Cy3 Gel 04 Cy5 Cor Cy5 Min Gel4dia Gel4 xml 2500 0 A 100 P 100Y 1C Whole Image Pick gel Unas Dia p sog 0 A 100 P100 V 1C Whole Image E Processing DIA Batch List BVA Batch List
109. able view Area Area as expressed in pixels within a spot boundary Peak height ratio Ratio of the normalized peak heights of a pair of spots from a spot map pair A value of 2 0 represents a two fold increase while 2 0 represents a two fold decrease whilst a value of 1 00 represents an unchanged spot Spot radius Refers to the radius that the spot would have if it was circular and had the same area Excluded Indicates whether spot has been excluded DeCyder Differential Analysis User Manual 18 1173 16 Edition AA XML Toolbox Confirmed Indicates whether a spot has been manually confirmed by the user Confirm Status Indicates whether the user has confirmed protein as real Auto Confirm Assignment Refers to the spot status after application ofthe exclude filter Spots are either designated as excluded or included _ DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 137 XML Toolbox 138 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA LWS Integration 8 8 LWS Integration 8 1 Overview The XML Toolbox has been designed to enable the smooth transfer of files containing pick lists from DeCyder Differential Analysis Software to Ettan LWS software product number 18 1164 06 and the transfer of protein identification results from Ettan LWS software after MALDI mass spectrometry back into DeCyder Differential Analysis Software Scan gel in Typhoon Variable Mode Imager Analyse
110. accession number for database linking Appearance Indicates the number of Spot Maps in which the selected spot appears and the function these Spot Maps have Example 20 20 A M means that the spot is present in 20 out of 20 Spot Maps Furthermore the Spot Maps are all assigned to be used in statistical analysis The spot is also present on the master image T test p value calculated using the Student s T test Average ratio Average ratio between the groups selected in the protein statistics dialog box 1 ANOVA p value calculated using One way ANOVA statistical test 2 ANOVA p value calculated using Two way ANOVA Condition 1 statistical test with respect to Condition 1 2 ANOVA p value calculated using Two way ANOVA Condition 2 statistical test with respect to Condition 2 2 ANOVA Interact p value calculated using Two way ANOVA statistical test with respect to interactions between conditions 1 and 2 Picked Indicates whether the protein spot has been assigned for picking Table continued on next page DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN Pick spot volume Displays the volume of a spot designated for picking after spot detection and background correction This value can be used as an indication of whether a spot can be identified by mass spectrometry Function Proteins assigned Protein of Interest are marke
111. ace ooccccccooooccccnoooonanoconononanonocinonos 177 11 4 4 Exporting Spot Map Dala ion tii 179 11 5 Creating the BVA workspace cccnocooccnccccnoooncnnnnnononocononononnnccnnonos 180 TES Selci Del iaa ee 180 11 5 2 ESTA A cad iea iaa aiaa 182 11 5 3 Experimental group assignment ccooocccccnnnooocnnccnnooananocinncnnns 183 LILEDA Landmarkihp doi da h 184 11 6 Assessing Matching cccccococccccnnococcnnnnooonnnnnnnnnnnncnnncnnnonanonnnono 186 11 7 Post matching landmarking cocooooccccconococnncnnononncononnononancnoninnna 189 11 8 Statistical analysis ccoococccccnnccoococonoconononononononnnnnonoononanonnnnnons 190 11 9 Assigning spots as proteins of interest oooooooccnnccccccccccononnccnono 193 119 1 Selecting proteins Of interest oooooconocicnccoocnncccnoconananonononos 193 11 9 2 Spot confirmation in Protein Table 195 119 3 Exporting the Pick List and physically excising spots AA A yes 195 12 Tutorial Ill Processing the Preparative Gel and Generating a Pick List LATOBDECVO ad a A Rae Ne de es 197 T22 Ove eW nenda eote e ote thie old Ate ves teat 198 12 3 Experimental design cmos er ataa i A redrai Tis 198 12 3 1 Identifying protein spots on SYPRO Ruby stained gel 199 12 3 Spot detecto si e E 200 12 3 3 Assigning an area of interest ocooooccoccccnoconcccconoconananoconcnnnn 203 12 3 4 Gel artifact removal ecreis ii a eaaa ae 206 12 4 Matching to analytical gelS oooooococccccncoooocccc
112. ach image and express these values as a ratio thereby indicating changes in expression levels by direct comparison of corresponding spots This ratio parameter can be used in small scale experiments to directly evaluate changes between two labelled protein samples Alternatively the ratio can be used for protein spot quantitation of a sample against an internal standard to allow accurate inter gel protein spot comparisons see Chapter 1 Once spot maps incorporating an internal standard have been analyzed in the DIA module the spot data can be used in DeCyder Differential Analysis Software BVA for accurate quantitative inter gel studies Generally when multiple gel analyses are performed only the spot detection and quantitation algorithms are utilized in the DIA module Data is then transferred to the BVA for inter gel analysis Data can be saved and re opened in a DIA file format from within the DIA module The DIA module can also export for both Ettan Spot Picker or Ettan Spot Handling Workstation Data can also be exported in an XML format for either multi gel analysis in DeCyder Differential Analysis Software BVA querying in DeCyder Differential Analysis Software XML toolbox or copying and pasting into applications such as Microsoft TM WORD and EXCEL DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Def DIA Differential In gel Analysis Module 28 DIA Graphical User Interface DeCyder Differential Analysis Soft
113. al Variation Analysis Module 78 4 8 5 Student s T test amp Average ratio Average ratio Log standardized protein abundance is the only variable analyzed in the Student s T test within DeCyder Differential Analysis Software BVA module The degree of difference in the standardized abundance between 2 protein spot groups is expressed as the average ratio The average ratio value indicates the standardized volume ratio between the two groups or populations Values are displayed in the range of to 1 for decreases in expression and 1 to for increases in expression Values between 1 and 1 are not represented hence a two fold increase and decrease is represented by 2 and 2 respectively not 2 and 0 5 Note The average ratio parameter is displayed in this manner but the statistical analyses are based on the log of the true ratio measurement Student s T test Student s T test often known simply as the T test is one of the most commonly used of all statistical tests The Student s T test is used to test the hypothesis that a variable differs between two groups The Student s T test performed is an equal variance two tailed test therefore direction of change i e increases and decreases in the standardized abundance parameter are considered The T test requires a minimum of two data points in each of the two groups Greater statistical validity can be achieved using larger number of replicates It is recommended th
114. allation is given if this is not suitable browse for an alternative path Click Next to install the software to the selected path DeCyder Differential Analysis Software Setup will install DeCyder Differential Analysis Software in the following folder To install to this folder click Next To install to a different folder click Browse and select another folder Destination Folder C Program Files Amersham Biosciences DeCyder Browse InstallShield ZEN DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 25 ES Computer requirements and installation A The HASP key and DeCy der Differential Analysis Software will now be installed When setup is complete click Finish Note If DeCyder Differential Analysis Software is supplied pre installed on a computer no further software should be installed 26 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA DIA Differential In gel Analysis Module 3 DIA Differential In gel Analysis Module 3 1 Overview DIA Processes DeCyder Differential Analysis Software DIA processes images from a single gel performing spot detection and quantitation The DIA module algorithms detect spots on a cumulative image derived from merging up to three individual images from an in gel linked image set This co detection ensures that all spots are represented in all images processed The DIA module algorithms then quantitate spot protein abundance for e
115. alysis Spot map comment User defined comment on selected spot map Spot map function Spot map function in DeCyder Differential Analysis Software BVA M A T and P represent master analysis template and pick respectively Image data Volume Sum of the detected pixel values above background within a spot boundary Peak height Pixel value at the X Y position of the spot This represents the highest pixel value within the spot boundary Normal volume Normalized volume obtained by dividing the calculated volume for each included spot by the center of volume of the corresponding spot map Gel data Volume ratio Ratio of the normalized volumes of a pair of spots from a spot map pair A value of 2 0 represents a two fold increase while 2 0 represents a two fold decrease whilst a value of 1 00 represents an unchanged spot Spot comment User defined comment on selected spot Picked status Indicates whether the protein has been assigned for picking X pos Position of a spot peak along the horizontal axis of the spot map Y pos Position of a spot peak along the vertical axis of the spot map Protein ID Unique protein identifier that can be used to search databases providing it is in the correct format for the specific database Area Area as expressed in pixels within a spot boundary Match confidence Indicates the type of match achieved i e Unmatched Auto Level1 Auto Level2 Manual or Added to Master Spot Map DeC
116. alysis User Manual 18 1173 16 Edition AA Continued from last page Load the preparitve gel as the primary Is a preparative Yes gel present image Click OK No Click Cancel le Click Yes when prompted to assign this gel as the Pick gel When prompted to set a BVA workspace select Yes Assign attributes to each spot map Define the statistical test s required Y Select the Protein of Interest check box Y Enter the criteria for proteins of interest filtering Y Name amp stipulate the destination of the resulting BVA file DIA file for each y gel Select Run Process run to start processing immediately or delay as desired XMLfile for each gel BVA file for whole experiment DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 239 240 A 2 Flow Diagram 2 BVA file for whole experiment Open BVA module amp select File Open Workspace to open BVA generated in Batch Proocessor Y In Match Table mode check for accurate matching of all spot maps by observation of match vectors 3 D view and image view data Re match landmarked images Process Match Match Pending and Landmarked Is the gross atching accurate Yes y In the Protein Table tab ofthe Properties dialog box select the Protein of Interest check box to ens
117. arative gel in the DIA module is identical and is described first 5 2 Spot detection on the preparative gel The preparative gel gel picked from is prepared and scanned as described in Ettan DIGE System User Manual The preparative gel is normally prepared after the analytical gels have been investigated Therefore it is often necessary to perform spot detection independently on the preparative gel This is done as described in section 3 3 1 employing the single detection algorithm DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 107 ER Spot picking 108 The DIA XML file for the preparative gel must then be exported File Export Spot Maps so that it can be loaded into the BVA module for matching against analytical gels The XML file is exported after the identification of reference markers if the identification is performed in the DIA module 5 3 Identification of reference markers The reference markers placed on the preparative gels seen as circles on the images act as reference points for Ettan Spot picking robot Identification of the reference markers can be done in either the DIA or the BVA module the process is identical in both cases The reference markers can be detected automatically within the DIA module during the spot detection process Once a fluorescently post stained gel image is loaded into the DIA workspace select Process Process Gel Images to open the Process gel images window E
118. are true protein spots or non protein spots such as a particle of dust that has not been removed by the Exclude Filter function This can be determined by using the Image View and the 3 D View Click Confirm if the spot is a genuine protein spot The next spot in the list will now be automatically displayed If it is non protein spot select Exclude in the Spot Assignment toolbar and then click Confirm I Pick IF PTM 3 Confirm J Protein of Interest D Exclude Continue with this process until you have confirmed all the relevant spots 10 6 3 Exporting the Pick List and physically excising spots from the gel To excise the spots from the gel a separate preparative gel should be used This is then matched to this analytical gel set in DeCyder Differential Analysis Software BVA module and the protein of interest status is transferred to the relevant matching spots on the preparative gel The pick list is based upon these proteins The pick list with x y co ordinates of spot positions is then exported based on the preparative gel The pick list with the physical preparative gel is then taken to Ettan Spot Picker or Ettan Spot Handling Workstation for spot excision and later analysis by mass spectroscopy Generating a pick list is covered in more detail in Tutorial TIT 172 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial Il Employing an internal standard to Analyze Protein Changes Oy 11 Tutorial Il Employing a
119. at the largest possible number of biological replicates are performed for optimal validity However if biological replicates are not possible gel replicates can be used for the purposes of the statistical analysis In these instances it is recommended that gels be run at least in triplicate hence each group has a minimum of three data points Null hypothesis The Student s T test null hypothesis is that there is no change in the protein abundance between experimental groups i e the average ratio between two groups is 1 Therefore the T test p value seen in the T test column of the protein table represents the probability of obtaining the observed data if the two groups have the same protein abundance For example if the T test p value between two groups is 0 01 then the probability of obtaining the observed difference in protein abundance by stochastic variation alone is 1 in a 100 Protein abundance differences are generally assumed to be statistically significant when p lt 0 05 However a critical value of 0 05 would mean that 5 of the data points would be expected by stochastic events alone DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN A e g 50 spots out of 1000 tested It is therefore advisable to review the critical value applied to the data Due to the small amount of experimental variation within Ettan DIGE system and subsequent DeCyder Differential Anal
120. ave 65 536 shades of gray 0 being black and 65 535 being white Table B 2 Bit depth and their corresponding number of different combinations available Depth Example Combinations 1 bit O 2 2 bit 10 4 4 bit 0110 6 8 bit 01010011 256 12 bit 011010011101 4096 16 bit 1011000100101101 65536 e Pixel frequency histogram The frequency histogram refers to the frequency representation of different shades of gray or color in the image A frequency histogram displays the number of pixels representing each grayscale or color value Frequency histograms most commonly represent grayscale images and have many uses the histogram may reveal an under or overexposed image too many pixels with values close to 0 or too many with values close to 255 respectively and the histogram can be manipulated to change the image frequency values can be deleted and upper and lower thresholds can be set B 5 Image dimensions e Resolution Image resolution is often confused with pixel dimension and refers to the number of pixels displayed per unit length of an image Pixel resolution is the fineness of the divisions into which the scanner partitions the image When the divisions are extremely small the scanner is said to have high resolution when the divisions are course the scanner has low resolution DeCyder Differential Analysis Software requires images which have a pixel resolution of 100 pm Anything less does not
121. between spots of the same protein This parameter is referred to as the abundance in the BVA module DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 255 256 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Appendix D Experimental design and set up examples In this appendix there are examples of complex experimental designs and statistical analyses There are descriptions of both a paired testing and Two Way ANOVA which include details of the experimental design setting up the statistical analyses and interpretation of example proteins D 1 Example of a paired experiment Experimental objective An experiment is designed to investigate changes in protein expression caused by a drug treatment after 24 hours in human subjects Experimental design Blood samples are taken prior to drug treatment from five volunteers Blood protein was individually extracted and stored The drug was then administered to each individual blood samples were taken 24 hours later and protein was extracted Ten aliquot samples derived from the five individuals were taken and pooled to act as the internal standard The pooled standard was labelled with CyDye DIGE Fluor Cy2 minimal dye Pre treated and treated blood samples from each volunteer were independently labelled with either CyDye DIGE Fluor Cy3 or Cy5 minimal dye Equivalent amounts of labelled standard volunteer 1 pre treated volunteer 1 post treated were mixed and s
122. click OK to begin spot detection on the images The software then processes the two images and calculates a series of values for each spot on the two images The DIA analysis takes between 1 and 5 minutes depending on the specifications of the computer DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 221 Tutorial IV Fully automated identification of differentially expressed proteins Before going any further it is important to become acquainted with the main tool bar AE ESA SEE Create Workspace What s this Open Workspace Print Save Workspace y Properties ProcessGels All Views Pick Filter Table View Exclude Filter 3D View Areain3D Histogram View Rotate 3D Image View Zoom In Brightness C ontrast Zoom Out Fit to Window 4 When the DIA analysis has finished the screen below appears E Decyder DIA Control Treated dia Fie Edt View Process Help DSW ee 222 ee ng Primary Gel 02 Control Gy3 H Secondary Gel 02 Treated a nn Ai jax Volur gt Histogram selections Threshold mode 2 model S Threshold 2 41551 25 D 232553 Spot statistics Decreased 116 4 8 Similar 2235 91 6 Increased E 80 3 7 wiohistogram View Detected 2441 Included 2441 a Excluded 0 Log Volume Ratio Picked o Number of Spots Status Spot No Abundance Excluded Volume Ratio Picked Unconfirmed 2317
123. contain the required amount of information whilst anything more adds no further advantage to the image analysis procedure Resolution determines the area occupied by the images in conjunction with the pixel dimension and is a measurement of clarity or detail It can also refer either to an image file or the device such as a monitor used to display it The relationship between number of pixels and area DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 247 248 is commonly expressed by number of pixels per inch ppi the more pixels per inch the better the resolution Output print or display resolution is more commonly expressed in terms of dots per inch dpi The resolution of a scanned image is also expressed in dpi where the number of dpi is equal to the number of ppi i e an image scanned in at 300 dpi will give you an image resolution of 300 ppi Image file resolution and output resolution combine to influence the apparent clarity of a digital image when it is viewed The display monitor used will also influence apparent image quality The amount of resolution is often ruled by practical considerations e g the higher the dpi number the more information in the file and the greater the ability to enlarge a detail from that image If the principal life of an image is on screen e g an image for a web page as opposed to being printed out and if details for enlargement are not required from it a resolution of 100 dpi w
124. ctric point pl of all if i ift the proteins on the images select Process Calibrate pl and Mw or click the Calibrate pl and Mw icon The subsequent dialog box displays the pI and Mw values that have been manually entered into the protein table using the data control panel in the Protein Table mode These values are used as a base list to calculate the pI and Mw values for all the proteins Calibrate pl and Mw Calibration option pl Mw Linear C Linear Log Linear Log Linear Cubic Spline Cubic Spline The calculation will be based on following values Master No Protein ID Mw 4 2 00 5000 67 2 50 7500 129 3 00 10000 167 3 50 1250 289 4 00 15000 Calculate Calibration of the pl and Mw values can be user determined by selecting Linear Log Linear or Cubic Spline e Linear Creates a straight line between all values in the base list e Log Linear Similar to the linear function except that the values in the base list values are first logged Log 9 So the line is based on logarithmic values e Cubic Spline Creates a line using the cubic spline function which fits a curved line through all points in the base list The type of first dimension Isoelectric Focusing IEF strip influences pI calibration used For example a linear Isoelectric Focusing IEF strip Sey 92 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN
125. culate to perform the analysis DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 83 ED BVA Biological Variation Analysis Module Protein Statistics Type of statistical tests Independent tests normal Paired tests use sample ID Statistical tests I Average Ratio Student s T test Population 1 Population 2 Dption s Option s 3 Members 3 Members IV One Way ANOVA between different groups FT TwoWay ANOVA between Dose and Time Calculate Cancel Help The One Way ANOVA p values will then be displayed in the protein table As with the T test analysis spots can be ordered by significance by clicking on the 1 ANOVA column heading in the protein table Specific requirements All groups that have at least two members will be included in the calculation To analyze a subset of groups those that are to be excluded have to be de selected as analysis in the Spot Map function tab see section 4 5 2 84 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN 4 8 8 Two Way ANOVA Overview Two Way ANOVA calculates the significance of the difference between groups with the same condition 2 and different condition 1 value Two Way ANOVA Condition 1 and the other way around Two Way ANOVA Condition 2 The Two Way ANOVA analysis also calculates a significance value of the mutual effect of the two factors Two Way ANOVA Interaction
126. d secondary images are changed using the pull down menu in the Image View title bar 40 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA DIA Differential In gel Analysis Module Histogram Selection The Histogram Selections toolbar contains two drop down menus and two information boxes Scatter Parameter and Threshold mode Scatter Parameter The pull down menu can be used to plot either maximum peak height area maximum slope maximum volume displayed on the right Y axis of the histogram against log volume ratios Threshold mode Thresholds are user defined values that enable highlighting of spots that differ between the primary and secondary image Consequently the threshold functionality is predominantly used when performing small scale experiments to determine expression differences in two samples run on a single gel A variety of values can be selected in the threshold pull down menu e Model S D standard deviation number of standard deviations based on the normalized model curve displayed in the histogram view Fold magnitude of volume ratios e Manual user defined magnitude of volume ratios entered in the threshold text box The threshold values are represented by vertical black lines in the histogram Spot boundaries in the Image View and data points in the histogram are automatically color coded to denote the spots that are below within or above the threshold values The abundance colum
127. d with an I in this column pl Isoelectric point of protein can be user defined or calculated Mw Molecular weight of the protein can be user defined or calculated Name User defined protein names Comment User defined notes on proteins Match Quality Morphological similarity metric describing deviation of internal standard spot to an average internal standard spot in protein set PTM Indicates that the protein has a post translational modification DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 99 ED BVA Biological Variation Analysis Module E The columns displayed in the protein table can be changed using the Protein Table Properties dialog box DeCyder BYA Properties Image View 3D View Graph View Database Colors Spot Map Table Match Table Protein Table Appearance Table Protein Table Filter I Protein of Interest I only I Proteins assigned as Pick only Table Column Order and Visibility Select columns to display and the column order by drag and drop Po Master No Status Protein ID Protein AC Name Appearance T test Ay Ratio 1 ANOVA 2 ANOVA Dose 2 ANOVA Time 2 ANOVA Interact Picked Pick Spot Vol Function pl Mw Comment Left Column lt a laa la K Right Column Restore to default order and selection Default Cancel Help Protein Table Filter Select check
128. d sample denoted in the gel image file name apparent in the dialog box title bar DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 229 Tutorial IV Fully automated identification of differentially expressed proteins 230 3 To assign a spot map to either a control or treated group the two group names must first be created Click on the Add button in the Experimental Design section of the dialog box and type the name Control Click OK Click the Add button again then type the name Treated click OK Item 2 Gel 01 Cy3 Control gel Spot Map Assignments Spot Map function IV Analysis IT Master Template IT Pick Experimental Design Group Unassigned Se Group description Add Group Condition 1 Please enter the group name Control Sample ID Once the two experimental groups have been created the remaining analytical spot maps can be assigned to their group sequentially using the Group pull down menu in the Spot Assignment dialog box followed by clicking OK The last spot map is the pick gel item 13 This gel does not belong to an experimental group and can therefore be left Unassigned Deselect the Analysis check box in the Spot Map Function part of the dialog box Click OK to complete the spot map assignment process DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial IV Fully automated identification of differentially expressed proteins A 13 5 3 Protein statistic
129. d spots Normalization Process that allows the direct comparison of spot data derived from different gels Null hypothesis Posits that there is no difference between variables being tested To reject it is to infer statistical significance Paired Data Data sets with an association between data point in every group Pick Filter Function that enables the user to designate spots for picking based on user defined parametric values Primary image Refers to the left hand gel image present in the image view of the DIA module and the spot map table protein table and appearance table modes of the BVA module In the Match Table mode of BVA module the primary image is the right hand image as the left hand image is taken by the master gel Protein ID Unique protein identifier that can be used to search Protein Table Area within a BVA workspace where all the statistical data from the analytical gels is located Protein Statistics Function in the BVA module that applies statistical tools to the protein data providing that an experimental design with an internal standard was used Repeated Measures ANOVA Statistical ANOVA test applied to paired data Scatter Parameter The spot data type used to display the spot ratios in the histogram This is shown in the right y axis on the histogram and can be maximum slope volume peak height or area Secondary image Refers to the right hand gel image present in the image view in th
130. d subsequent spot excision is conventionally loaded last Load the Pick gel as a primary image then click OK 9 Since the file name contains the text pick the software prompts the user to set this gel as the pick i e to use this gel for generating the co ordinates for the pick list Select Yes in this dialog box 228 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial IV Fully automated identification of differentially expressed proteins 10 When all the images have been loaded click Cancel The batch information is then entered into the DIA batch list and the user is prompted to set the BVA workspace Click Yes EN Batch Processor ai pia Di _ C AFiman Tutorial images v5 Temp Tutoiald Browse BVA Filename C Fiman Tutovial images v5 Temp Tutorial XML Di C AFiman Tutorial images v5 Temp Tutoiald Browse Pick Filename DIA Batch List if BVA Batch List Status Primary Label Secondary Label Tertiary Label Type DIA File XMLFile Estimated Pick ref Found Exclude Fiter Area of interest Pending Gel01 Cy2 Sta Cy2 GelO1Cy3Cor Cy3 Ge101 Cy5 Tre Cy5 Min Geltdia Gel 1 xml 2500 No 5 0 A 100 P 100 V 1 Whole Image Pending Gel02Cy2 Stal Cy2 Ge 02 Cy3 Tre Cy3 Gel 02 Cy5 Cor cy5 Min Gel2da Gel 2xml 2500 No 5 04 10 P 100 V 1C Whole Image Pending Gel 03 Cy2 Stal Cy2 Gel 03 Cy3 Cor cy3 Ge103 Cy5 Tre Cy5 Min Gel3dia Gel3 xml 2
131. de in graph view The options available determine the data points plotted on the graph By selecting the Spot check box all proteins spot data points associated with the user defined X axis parameter are shown Colored circles represent these data points The colors representing the groups can be assigned in the Experimental Design view Exp Design View a i is Bey BYA Experiment Gel 01 Cy3 Control gel Group Eon a 1JGel 02 Cy3 Control gel E Groupi Gel 03 Cy3 Control gel Ses E Group2 1 JGel 04 Cy3 Control gel pessiptor E Unassigned JGel 05 Cy3 Control gel Control PS FH Treated ee Condition2 Confirm Selecting the Mean Value Crosses check box results in the mean value of the data points associated with the user defined X axis parameter being displayed represented by a cross Lines connecting these mean values can be displayed by selecting the subsequent check box If either Groups or conditions are assigned to the X axis the Y axis values corresponding to the internal standard can also be displayed by selection of the Standard check box DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN Note The DeCyder Differential Analysis Software standardization process defines all protein abundance values associated with the internal standard to be 1 or zero if the log values are displayed For further details see Appendix C The legend check box al
132. e DIA module and the spot map table protein table and appearance table mode of the BVA module Slope Maximum gradient associated with the 3 dimensional attributes of a spot map pair DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 277 Spot Controls Allows user to control assign or input data to the workspace or individual protein spots Spot Map Table Area within a BVA workspace where all the spot map images and their associated assignments are located Spot Number Unique identifier for a spot in DIA or a spot set in BVA Standard Image Image relating to the internal standard from each gel Standardization Process of quantifying spot map data in relation to the corresponding standard spot map data Standardized Abundance Abundance relative to the standard image displayed as a ratio Student s T test Statistical test that assesses differences between two populations Threshold Mode Value above or below which spots are classed as being differentially expressed TIFF Image Flexible image format used to exchange files between platforms and software applications Volume Sum of the detected pixel values above background within a spot boundary Volume Ratio Refers to the ratio of the normalized volumes of a pair of spots from a spot map pair A value of 2 0 represents a two fold increase while 2 0 represents a two fold decrease whilst a value of 1 00 represents an unchanged spot Workspace Environm
133. e entitled Ecoli Control treated for picking in the folder Tutorial III in the Tutorial data files folder 3 Double click on the BVA file to open the workspace this may take a few minutes depending on the specifications of the computer This BVA file contains the experiment described in the experimental design section of this tutorial This file has been pre analyzed in the BVA module and several proteins that demonstrate a statistically significant change upon treatment have been given a Protein of Interest status and hence will require picking and subsequent identification Note If this tutorial is being performed directly after completing Tutorial II the created tutorial II workspace can be used instead of the pre analyzed BVA file The BVA interface consists of four different modes Spot Map Table ST Match Table MT Protein Table PT and the Appearance Table AT Each screen in the BVA interface possesses a similar layout consisting of e Image View Displays the gel images making up the experimental set with the currently selected spot shown in magenta e Table View Depending on the screen selected displays either information on the images used in the experiment different properties for a single spot across the gels in the analysis set or protein spot specific statistical values e 3 D View Representation of the selected spot in 3 dimensions e Graph View Graphical representation of the protein abundance data fo
134. e for preliminary investigation of protein changes 10 10 Tutorial I Using DIA module for preliminary investigation of protein changes 10 1 Objective This tutorial describes how to find protein abundance differences above experimental variation between a control and a treated protein lysate of bacterial cultures using DeCyder Differential Analysis Software DIA Differential In gel Analysis The procedure in this tutorial can be applied to any two protein mixtures lysates This experimental design can be used to rapidly identify proteins that show a substantial change in abundance from control to treated sample and is often used at the preliminary stages of an experiment The method is used to gain early information for the samples in question and to check that the experimental procedures are appropriate for the new samples The major drawbacks of this approach are that only one control sample and one treated sample are studied There are no gel replicates or biological replicates and therefore the biological relevance and statistical validity of an altered protein is impaired This experimental design only addresses differences above system variation see section 1 3 for different sources of variability Since there are no biological replicates the inherent biological variation within populations is not considered This is also true if the biological control and treated sample are pools of control and treated populations as this approac
135. e internal standard on the same gel to generate a ratio of relative protein levels Quantitative comparisons of samples between gels are made based on the relative change of sample to its in gel internal standard This process effectively removes the system gel to gel variation enabling accurate quantitation of induced biological change between samples Fig 1 2 The need to run gel replicates is also overcome reducing the number of gels required per experiment DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Introduction to DeCyder Differential Analysis Software er 0 o o 2 A Cy2 Internal Cy3 Sample 1 Cy5 Sample 2 Standard Os Cy2 Internal Cy3 Sample 3 Cy5 Sample 4 Standard System variation due to protein loss in Gel 2 Real induced change reduced protein abundance in sample 3 Fig 1 2 Analysis of samples 1 4 in the absence of an internal standard would suggest that the protein spot circled in red on gel 1 is absent in samples 3 and 4 However reference to the internal standard which is an identical pool of all samples run on both gels shows that this protein has not entered gel 2 This indicates that the observed absence of this protein spot in samples 3 and 4 is due to system variation e g gel distortions differences in first dimension focusing etc and not to sample differences Similarly analysis without the internal standa
136. e is information in either the Protein ID or the Protein AC column of the Protein Table Upon selecting a protein only databases that have placeholder strings that fit the information entered for the protein will be available To verify that the address is entered correctly select a protein in the Test pull down menu and click the Test button If the address is correct the browser should open and information on the test protein should be shown DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN Example Database SwissProt AC or ID search Address with substitution string http www expasy ch cgi bin get sprot entry BVA_PROTIDAC Protein AC Human myoglobin P02144 The web browser will open at the address http www expasy ch cgi bin get sprot entry P02144 If a system for identifying proteins other than AC or ID is being used text can be entered manually or pasted into the test place holder string box Accessing databases Ensure that the required database is selected in the Properties window by clicking on the Properties icon and selecting the Database tab Select the desired database s from the Database Selection list then click OK To access a database right mouse click the protein to be queried in the Protein Table then select the database If both Protein ID and Protein AC labels are available a choice of the label to be used is given The web brow
137. e level of experimental variation The protein abundance variation calculated in DIA indicates the degree of similarity between the protein spots from two images from the same gel Ideally there should be no difference between the proteins from the two control control gel images because the same sample is being run on the same gel However due to different experimental factors for example pipetting variation there is some intrinsic variability The data analysis is performed in three parts using the DeCyder Differential Analysis Software DIA module e Analysis of control control gel e Analysis of control treated gel e Creating a spot pick list 10 4 Analysis of control control gel 10 4 1 Selecting gel images 1 Double click the DeCyder Differential Analysis Software DIA icon on your desktop to open the DIA module DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial Using DIA module for preliminary investigation of protein changes 10 E V Select File Create Workspace File Est View Process Help Create Workspace Ctrl N Open Workspace Ctrl O Save Workspace Ctrl4s Save Workspace As 3 Select Double Detection then click OK Type of Detection Single Detection one image Double Detection two images Triple Detection three images Cancel 4 Browse to locate the folder entitled Tutorial I in the Tutorial data files folder Double click on the image Gel 01 Contro
138. e number of spots have been manually excluded In the DeCyder Differential Analysis Software DIA environment the expression ratios are displayed differently in the table than in the histogram The histogram ratio representation Ri is illustrated above while the expression ratio E presented in the table is calculated according to E 10 for R gt 0 E 1 10 for R lt 0 iv This results in a value less than 1 for under expressed ratios and a value larger than 1 for over expressed ratios Equal ratios are expressed as a ratio of 1 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 253 254 C 2 BVA Normalization If an internal standard is not used in the experimental approach DeCyder Differential Analysis Software can normalize spot maps facilitating the comparison of spot volumes on different gels The normalized volumes for a set of spots for a protein are then expressed as a ratio of the weakest spot This parameter is referred to as the abundance This approach is not recommended as inter gel quantitative comparisons are not as accurate as the quantitation using an internal standard The steps involved in the BVA normalization process are described below 1 Calculate spot volumes Based on the result of the spot detection algorithm the volumes of the detected spots are calculated and compensated for the appropriate background level Background is subtracted on a spot specific ba
139. e rapid access to both local and web based databases through Protein IDs and Accession Numbers AC This functionality is designed to aid further study of identified proteins using information from existing databases A list of web based databases are provided as default in DeCyder Differential Analysis Software The linking uses the default web browser to interact with the databases The Protein ID or AC is substituted into the web address of the database On condition that the protein ID entered in the protein table is in the format that is recognized by the search algorithm of a database the user 94 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN can directly access a selected protein from BVA In DeCyder Differential Analysis Software BVA module it is possible to link proteins to information in databases Use the Database tab of the BVA module property pages to set up how Protein IDs and ACs are inserted in the web address of different databases 4 12 1 Adding databases To add a database to the default list of databases in BVA click on the Properties icon or select View Properties to open the Properties window then click on the Database tab DeCyder BYA Properties Spot Map Table Match Table Protein Table Appearance Table Image View 3D View Graph View Database Colors r Database selection SwissProt a
140. eck box is selected Click OK Item 1 Estimated no of Spots and BYA Processing x Estimated no of spots 2500 J7 Autodetect pickreference markers IV Include in BYA batch list DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 227 Tutorial IV Fully automated identification of differentially expressed proteins The filter parameters generated previously in DIA can now be entered in the Exclude filter dialog box Place a tick in each of the boxes on the left hand side of the different filter properties to activate the filter then enter the filter parameters generated in DIA in their respective boxes Click OK Item 1 Exclude Filter Spot Properties Use Area of Interest IT Slope Area of Interest x V Area y o X distance 7 Peak Height Y distance IV Volume lt 20000 The second set of images associated with the second gel now have to be entered in the batch processor 7 In the dialog box which now appears enter the primary secondary and tertiary gel images associated with the second gel in an identical manner to the first gel Click OK The remaining gel processing parameters such as spot detection and the exclude filter variables entered for the first gel is automatically applied to the second gel Continue loading all the images in this sequence until all the images from all four analytical gels have been loaded 8 The preparative gel used for generating the pick list an
141. ecting File Export Workspace The DeCyder Differential Analysis Software XML file can then be opened in the XML Toolbox for further analysis Pick list data can be exported as a text file by selecting File Export Picking List From Primary Spot Map or Pick Spot Map The text file can then be utilized by Ettan Spot Picker DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Spot picking E 5 Spot picking 5 1 Overview DeCyder Differential Analysis Software provides the user with the functionality to generate a spot pick list which assigns proteins for picking by Ettan Spot picking robot The generation of the pick list in DeCyder Differential Analysis Software involves the following processes e Assigning proteins of interest which warrant identification on the analytical gels i e gels analyzed during experiment e Spot detection on the preparative gel gel prepared for picking see section 3 3 1 e Identification of reference markers on the preparative gel e Matching analytical and preparative gels e Confirming matches between analytical and preparative gels e Assign the proteins of interest as spots for picking e Confirming spots for picking e Exporting a pick list There are two principle means of generating a pick list within DeCyder Differential Analysis Software The method adopted is dependent on the size of the experiment and the desired picking criteria For both methods processing the prep
142. ection 4 6 2 Landmarking may be required to accurately match preparative gels see section 4 6 1 5 5 Assigning Spots for Picking As can be seen from the previous section a BVA workspace can be created using either the DIA module or the BVA module to identify a set of proteins as protein of interest in a workspace containing a preparative gel that has been matched to the analytical spot maps The preparative gel should then be assigned as the Pick Spot Map using the function check boxes in the Spot Map Table mode a y Function for Spot Map SYPRO gel iv Analysis A I Master M Template T The spot map Pick status denotes that the co ordinates for generating the pick list are based on the designated preparative gel It is recommended that the spots assigned as POI are visually inspected to remove those not suitable for picking and subsequent analysis This can be performed in the Protein Table mode of the BVA module The Protein Table must initially be organized so that POI proteins are exclusively displayed in the Protein Table This is accomplished by selecting the Protein of Interest 1 only check box in the Protein Table tab of the Properties dialog window DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Spot picking ea DeCyder BVA Properties Image View 3D View GraphView Database Colors Spot Map Table Match Table Protein Table Appearance Table Proteja LabloF IV Protein of
143. ed using the DeCyder Differential Analysis Software DIA Differential In gel Analysis module Gels are processed sequentially in the DIA module 11 4 1 Selecting gel images 1 Double click the DeCyder Differential Analysis Software DIA icon on your desktop to open the DIA module When the user interface appears select File Create Workspace File Edit View Process Help Create Workspace Ctri N Open Workspace Ctrl 0 Save Workspace Ctrl s Save Workspace As 2 Select Triple Detection then click OK Type of Detection C Single Detection one image Double Detection two images Triple Detection three images Cancel 3 Browse to locate the folder Tutorial II in the Tutorial data files folder Note All the images are named in the style Gel ON Cy X Standard Control or Treated DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 175 ED 1 II Employing an internal standard to Analyze Protein Changes AA Select Gel 01 Cy2 Standard then click Open to load the Primary Gel Image Load Primary Gel Image Tutorial I ly J e EB My Recent Gel 01 Cy3 Control Documents E Gel 01 Cy5 Treated gel E Gel 02 Cy2 Standard gel E Gel 02 Cy3 Treated gel E Gel 02 Cy5 Control gel E Gel 03 Cy2 Standard gel E Gel 03 Cy3 Control gel E Gel 03 Cy5 Treated gel la Gel 04 Cy2 Standard gel E Gel 04 Cy3 Treated gel E Gel 04 Cy5 Control ge
144. ee section 4 12 or DeCyder Differential Analysis online help DeCyder Differential Analysis User Manual 18 1173 16 Edition AA LWS Integration eCyder BVA Ettan LWS DeCyder XML Tools 1 0 bva File Edit View Process Help DE sarna o F He 22 A Primary Gel 01 Cy3 Contro y Master No 1991 Protein ID gi 1473791 Confirmed Confirmed Confirmed Confirmed Confirmed Confirmed Confirmed MLERL83IIANARANRAS Master No 1991 Pick Pos 686 917 686 917 Master No 1991 Pick Pos 566 951 566 951 da Erol 8 pl 520 Pick IT PTM 4 A M13174 phosphoribosylpyropho i ans l Vd Mw Da I Protein of Interest Focus Protein Table Fig 8 8 Example of results in DeCyder BVA after import and database query DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 153 ED LWS Integration 154 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorials Introduction 9 9 Tutorials Introduction 9 1 DeCyder Differential Analysis Software structure The DeCyder Differential Analysis Software suite is made up of four user interfaces DIA Differential In gel Analysis Protein spot detection and quantitation on images from the same gel BVA Biological Variation Analysis Matches multiple images from different gels to provide statistical data on protein expression levels Batch Processor Fully automated image detection and matching of multiple gels without user interaction
145. efficiently obtained by ordering the table according to each parameter in the filter To order the table according to Max Slope click once on the Max slope header on the table so that the largest maximum slope is at the top of the column if the smallest maximum slope is at the top click again Max Slope Area Max Peak Max Volume Click on the first spot in the Table View and observe the spot in the 3 D View The 3 D View clearly shows if the detected spot is a dust particle rather than a protein spot Working down the table leads to spots with smaller and smaller Max Slope values After a certain value the spots change from gel artifacts to real protein spots The max slope value of the artifact immediately before the real protein spot in the table is the value entered into the slope parameter of the filter The same procedure can be applied with the Area Peak Height and Volume The only difference with finding these parameters is that the table has to be ordered so that the smallest value is present at the top of the table before scrolling down If triple detection has been employed it may be necessary to toggle between the three images to ascertain more accurately the exclude filter parameters After the filter parameters have been established select Process Exclude Filter to display the Exclude Filter window and enter the values determined click OK The Exclude Filter will automatically remove DeCyder Differential Analysis User
146. el 03 Cy2 Standard g6 DIGE Min Cy2 2246 1679 A Standard 11 Matched Gel03 Cy2 Standard g DIGE Min Cy2 2455 1855 A Standard 13 Matched Gel 04 Cy2 Standard g7 DIGE Min Cy2 2422 1654 A Standard 15 Matched Gel 04 Cy2 Standard g8 DIGE Min Cy2 2323 1690 A Standard 17 Matched Gel 05 Cy2 Standard g DIGE Min Cy2 2339 1674 A Standard 19 Matched Gel 05 Cy2 Standard g10 DIGE Min Cy2 2138 1696 A Standard 4 Matched Gel 01 CyS Treated ge DIGE Min Cy5 2564 2564 A Treated BA treated 1 8 Matched Gel 02 Cy5 Treated ge DIGE Min CyS 2529 1843 A Treated BA treated 2 12 Matched Gel 03 Cy5 Treated geb DIGE Min CyS 2455 1855 A Treated BA treated 3 16 Matched Gel 04 Cy5 Treated geB DIGE Min CyS 2323 1690 A Treated BA treated 4 20 Matched Gel 05 Cy5 Treated geliO DIGE Min CyS 2138 1696 A Treated BA treated 5 2 Matched Gel 01 Cy3 Control gel 1 DIGE Min Cy3 2214 1865 A Control No treatment 1 6 Matched Gel 02 Cy3 Control gel3 DIGE Min Cy3 2204 1635 A Control No treatment 2 10 Matched Gel 03 Cy3 Control gel 5 DIGE Min Cy3 2246 1679 A Control No treatment 3 14 Matched Gel 04 Cy3 Control gel 7 DIGE Min Cy3 2422 1654 A Control No treatment 4 18 Matched Gel 05 Cy3 Control gel 9 DIGE Min Cy3 2339 1674 A Control No treatment 5 Note A set of images from the same gel will have the same number of spots since the DIA detection algorithm is designed to detect the same number of spots on images from the same gel In the Function column all the images are by defaul
147. en treated Since the gel to gel variation in this system is low gel replicates are not compulsory if biological replicates are available 3 Aliquots from each sample are taken and pooled to prepare a standard sample All three sample types standard control and treated are labelled with an appropriate CyDye DIGE Fluor minimal dye Cy2 Cy3 or Cy5 dye as described in the table overleaf Each sample is then applied to the gels DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 173 ED II Employing an internal standard to Analyze Protein Changes 174 4 The samples are separated within 4 gels by 2 D electrophoresis Three images are produced from each gel by scanning at appropriate wavelengths for each of the three fluors The recommended scanning instrument for this is the Typhoon 9000 series Variable Mode Imager 5 The images from the first gel are loaded into the DIA module 6 Spot detection and calculation of the spot properties are performed for all images from the same gel 7 Protein spots are normalized using the in gel linked internal standard 8 XML formatted data for the first gel is exported for subsequent matching and analysis in the BVA module 9 This XML data for the first gel and prepared XML data from the remaining gels are loaded into the BVA module 10 Spot maps images are assigned to groups and the experimental design is set up in the BVA module 11 All gels or those gels that require it to aid
148. ent where all the experimental data is stored XML Extended Markup Language Structured universal tagged language XML Toolbox Shell housing modules used for the extraction of data from DeCyder Differential Analysis Software XML files in the form of tab separated text or web tables 278 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA pner Index Numerics SED VIEW a et a 28 36 56 A ADUNGANCE aria 255 AddrattribUtE iia lo la ike 142 A aie i Wa een Mesias ees 95 AACOIOSSOS se device bina idas 3 Analysis image tarso olaaa daa seed eset Suva gele b ber ee 58 O cence ee a e be am ne utes 55 102 ANOVA dt ad 81 MENWAYS inici 75 83 98 WOW dedos 75 85 98 TWO WAY example oooooocccccnococcnnncnononcnnnonononnnanonoconananos 86 261 Appeara NCE eines done un aia dada 98 Appearance Table oconcococcncnococcnnnncnononnnononononnnnncnnonnnnanonnnnnnos 101 Area Ot Interest tween ati neti ole a 43 AWUITAGE Let id tia 44 Assign picking references cooocccnoncccnnnonanannnnonananananannnnnnnncnn nos 108 Ati DULES Coapa inet ee 142 AVErage ratio a vo en 78 B BaGkerouimnd tri id ta el 249 A A ech tide ten 121 Batch processor isnec oe aa 121 157 226 A ees sheet hcg Os tee setae E AEREE 51 156 BVA Module ari a tens plaice heirs als ea ats 51 c Calculate MW and PI aeee rete at aeee eate taenia etei mee AEA babak 92 Center of VOlUM star O ales O are ur 254 A A a atte adel AL 31 Computer req
149. eps for processing experiments that employ an internal standard including data analysis and pick list generation The workflow is split into three separate components which represent three phases of the analysis process e Flow diagram 1 describes the steps in preparing and processing the gels in the Batch Processor e Flow diagram 2 describes the steps in reviewing the proteins of interest in the BVA module e Flow diagram 3 describes the steps involved in processing a preparative gel then generating a pick list DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 237 A1 Flow Diagram 1 start Open Batch Processor amp select File New Batch Load the primary amp secondary 8 tertiary if present images from the first analytical geltobe processed Click OK Select the appropriate CyDye DIGE Fluor for each spot map Stipulate an appropriate file name for the results files Click OK It is recommended that the number 1 is appendedtothe file name of the first gel Enter the esitmated number of spots Seled the Include in BVA batch list check box to prompt inter gel processing Leave the exd ude filter check boxes de selected Is this the last analytical gel Load the primary image and secondary amp tertiary if present images Click OK No Yes Continued on next page 238 DeCyder Differential An
150. er to section F 1 for ordering details of the Scarce Sample Labelling kit A protein sample labelled with any of the CyDye DIGE Fluor dyes will migrate to the same position on a 2 D gel This permits the multiplexing of two or three samples within the same 2 D gel allowing the inclusion of an internal standard see section 1 4 Gels are scanned using the Typhoon Variable Mode Imager generating overlaid multi channel images for each gel Images can then be analyzed using the DeCyder Differential Analysis Software which contains novel algorithms for co detection of multiplexed gel images and has been specifically developed for use with Ettan DIGE system see section 1 4 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Introduction to DeCyder Differential Analysis Software er Pooled internal Protein extract 1 Protein extract 2 standard label with Cy3 label with Cy5 label with Cy2 Label protein extracts D 3 6D using 3 CyDye DIGE Qu Fluor minimal dyes Mix labelled extracts Y 2 D separation A A Image gel with Typhoon Variable Mode Imager AA Image analysis and data quantitation using DeCyder Differential Analysis Software Fig 1 1 Scheme showing the workflow for Ettan DIGE system The benefits offered by Ettan DIGE system are e Accurate quantitation and statistical analysis of
151. erest will be automatically removed when the Exclude Filter is applied The area of interest function is only operational when the Exclude Filter is executed If an area of interest was set before detection all the spots on the image will still be detected even those outside the area of interest 2 To set an area of interest click on the Fit to window icon on the tool bar to fit the gel images to the Image View Fh Click on the Image View icon on the tool bar to have a full screen view of the gel images Click on the Properties icon and select the Spot Display tab in the window that now appears De select Similar Increased and Decreased and Click OK to remove the spot boundaries displayed in the Image View de selecting the spot boundaries on view is optional but helps the user to see the gel more clearly DeCyder DIA Properties 3D View Tableview Colors Workspace Spot Display Image View Included spots Similar Decreased Increased I Excluded spots IV Picking references and pick locations Picked spots Cancel Apply Help Select Edit Define Area of Interest and using the rectangular target pointer drag the mouse to draw a rectangle on either of the gels taking care to DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 43 DIA Differential In gel Analysis Module 44 exclude edge artifacts The area of interest is automatically defined on the other image s Click on the
152. essor and import preparative gel XML file Assign the preparative spot map asthe Pick spot map Y Match preparative spot map to Master spot map Landmark in Match Table mode if necessary Continued on next page DeCyder Differential Analysis User Manual 18 1173 16 Edition AA A 3 241 A 3 A Continued from last page Select the first Protein of Interest in the Protein Table Y Check the matching between the prepartive and Master spot maps In Match Table mode use the Add Match or Break Match functions to add amend matches Isthe matching accurate Yes Y Assign Pick status tothe Protein of Interest Edit the pick location if necessary Is there another Protein of Interest Select the next Protein of Interest Yes No y Generate a pick list Select File Export Picking List From Pick Spot Map stop 242 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Appendix B Understanding the digital image This Appendix deals with the acquisition and definition of the digital image It explains some of the more common terms used in association with digital images and gives a brief insight into the GEL file format which is the Typhoon imaging systems default format B 1 Image acquisition In order for any digital computer processing to
153. expressed as unchanged i e log volume ratio of 0 The modified spot ratios are calculated in this step according to DeCyder Differential Analysis User Manual 18 1173 16 Edition AA LEE nA R log10 V2 V1 iii This parameter is termed standardized log abundance in the BVA module and is used for the statistical analysis 6 Re calculate data histogram The modified ratio values are then combined to form a new histogram of the same resolution as the previous one The normalized model histogram and data histogram are viewable in the histogram view of the DIA module represented by the blue and red lines respectively 7 Calculate data standard deviation The standard deviation of all the spot volume ratios according to iii is calculated This gives a rough indication of the spread of the data set Additional comments on spot normalization For this normalization procedure to be accurate the number of spots needs to be quite large It is recommended that more than 100 spots are present in the normalization However the more valid spots that are present the better If the number of spots to normalize is lower than 50 a model curve is not calculated In this case the position of the tallest histogram peak is used as C for normalization in ii When spots in the workspace have been manually excluded the workspace should be re normalized based on the new set of included spots This becomes exceedingly important if a larg
154. f the experimental design used for a simple three color experiment using control and treated groups each containing four individuals Gel Cy2 Cy3 Cy5 1 Pooled internal standard Control A Treated B 2 Pooled internal standard Control B Treated C 3 Pooled internal standard Treated D Control C 4 Pooled internal standard Treated A Control D Table 1 1 Using an internal standard for accurate measurement of protein abun dance changes between samples in an experiment for an experiment using gt 2 samples Each gel contains a pooled internal standard labelled with CyDye DIGE Fluor Cy2 minimal dye Four biological replicates A D have been included for control and treated samples that have each been labelled with CyDye DIGE Fluor Cy3 or Cy5 minimal dyes A greater degree of statistical confidence can be assigned to the experimental results by increasing the number of biological replicates employed Half of the control group are labelled using the CyDye DIGE Fluor Cy3 minimal dye half using the CyDye DIGE Fluor Cy5 minimal dye and similarly for the labelling of the treated group thereby confirming to best experimental practices For more detailed information about Ettan DIGE system and using an internal standard please refer to Ettan DIGE system User Manual code no 18 1173 17 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Introduction to DeCyder Differential Analysis Software er 1 5
155. format for use with either Ettan Spot Picker or Ettan Spot Handling Workstation respectively Click Save Set PickList Filename Save in Tutorial IV y e ex Ee My Recent Documents El Desktop My Documents My Computer My Network File name Pick list Places Save as type Text txt DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 233 Tutorial IV Fully automated identification of differentially expressed proteins A 135 6 Saving the batch workspace 1 To save the batch workspace select File Save As and enter a name for the workspace It is useful to enter a name that relates to the experiment Click Save 2 Set the BVA filename by clicking on the button marked Set on the side of the BVA filename box top right of screen Enter a filename that relates to the experiment e g E coli control treated This creates an empty file into which the BVA analysis output will subsequently be entered E DeCyder Batch Processor aaa File Edit View Process Help DIA Dir EAFimansTutoialimages v5 Temp Tutoriald Browse BVA Filename CAFimankTutorial images vo Temp Tutorial Set XML Dir EAFimankTutonal images vo Temp Tutorial d Browse Pick Filename C Fiman Tutorial images v5 Temp Tutorial Set Set Statistics Set Fiter DIA Batch List BVA Batch List l Image Type Label No of Spots Gel No Matched Function Group Group
156. g and MALDI mass spectrometry steps DeCyder Differential Analysis User Manual 18 1173 16 Edition AA LWS Integration 8 Sample Definition sample Definition Import Sample Definition Ettan 2D MS Project Selection ID tn425 Add to Project Project Name Y E Coli protein mapping Sample Type Protein y Sample Template DIGE El Comments Container Gel 2D El Picking gel stained with SYPRO Ruby E coli benzoic study Sample Attributes Name Value Concentration mg ml lo o Name tn425 Volume ul a o lcy2 unlabelled Backing material GLASS fia Cy3 unlabelled feys FA Backing thickness mm 3 2 Protein labelling unlabelled Image file spec LWS DIGElight LC301221 gel Browse Experiment E coli benzoic stu Evaluation file 2003 Scenario 1a LWS picklist xml Browse Finish Activity Clear Window Close Window Fig 8 5 Example of Sample Definition and entered values for the attributes when entering sample generated within Ettan DIGE system as a gel Methods Normally the standard methods included in Ettan LWS software and in Ettan MALDI ToF Pro can be used If non standard methods are to be used these methods have to be added e In Management Workbench System Method editor add staining method e In Scierra LWS select Spot Handling from the Workflow Graph In Tools Spot Handling Method Management Method Overview add Spot Ha
157. gels are expressed as a function of the internal standard allowing accurate quantitative inter gel comparative analysis To clarify some of the procedures involved in the DIA normalization a step by step algorithm description is provided 1 Calculate spot volumes Based on the result of the spot detection algorithm the volumes of the detected spots are calculated and compensated for the appropriate background level Background is subtracted on a spot specific basis by excluding the lowest 10 percentile pixel value on the spot boundary 2 Calculate spot ratios All the spot volume pairs are combined to create the set of ratio values for the entire spot map The ratios are calculated according to Rj log10 V2 V1 i Where Vj is the volume of spot i in the left or primary gel image and Vo is the volume of spot i in the right or secondary gel image The index i runs over all spots that are included in the analysis Spots that have status excluded are thus not part of the normalization The ratio R is also limited to the range 6 6 to avoid infinite ratios for zero volumes 3 Calculate data histogram The ratio values are then combined to generate a histogram plotting spot frequency against R i e log volume ratio The resolution of the histogram is 0 02 4 Optimize a model histogram curve A normal distribution is fitted to the main peak of the frequency histogram The tallest peak of the histogram C is
158. ges to be used as a template Master M One spot map can be assigned as the Master Spot Map in each workspace All other Spot Maps are matched to the Master When creating a new workspace the Spot Map with the largest number of spots is automatically set to be the Master Spot Map Template T One spot map can be assigned as the Template spot map in each workspace Protein ID Protein AC Name Comment pI Mw and Protein of Interest assigned to the spots of the Template Spot Map will be cross annotated to matching spots in all the other images Pick P This function should be assigned to a spot map if in DIA pick assignments have been made and the user wishes to transfer the pick status to corresponding spots in the BVA workspace see section 5 5 To assign the spot map functions select View Table View or click on the Magnify Table View icon to display the Table View only The table shows the images that have been loaded into the workspace and the number of spots that have been detected on them DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN Spot Map Table No Status Image Gel No Type Label No of Spots Matched Function Group Group Description Sample ID Comment 5 Matched Gel 02 Cy2 Standard g3 DIGE Min Cy2 2204 1635 A Standard 7 Matched Gel 02 Cy2 Standard gH DIGE Min Cy2 2529 1843 A Standard 9 Matched G
159. h is looking at averages but not variation associated with the different populations 10 2 Overview 1 An experimental design using two gels is set up control control gel and control treated gel 2 The control control gel is first analyzed in the DIA module From this an indication of overall system variation is determined by elucidating the fold change 95 of spots lie within e g 95 of spots on control control gel are within 1 5 fold 3 The control treated gel is subsequently analyzed in the DIA module As the control control gel gave a result that 95 are within X fold e g 1 5 fold then any spots with a fold change DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 159 10 1 Using DIA module for preliminary investigation of protein changes 160 DIA greater than X fold e g 1 5 fold has a 95 confidence that it is a difference between the control and treated sample above experimental variation 4 The spots greater than X fold on the control treated gel are looked at in detail These can be assigned with a protein of interest status and can be confirmed in preparation for spot picking Spot picking from a preparative gel is covered in tutorial III 10 3 Experimental design Two gels were loaded with bacterial lysates as indicated in the table below Gel Cy3 Cy5 1 Control Control Control Treated The control control gel gel 1 is analyzed to determine th
160. hat it fits exactly around the reference Magnifying the area of the gel around the reference marker by selecting the Zoom in icon may make accurate alignment easier Select Edit Edit picking References to exit this mode E When the first reference has been selected the same procedure is used to select the reference on the right hand side of the preparative gel image 5 4 Identifying Proteins of Interest Protein spots are first analyzed then filtered using various spot data parameters The spots are filtered on the basis of user defined criteria which result in the successfully filtered spot being assigned as a Protein of Interest POI These proteins of interest can be the evaluated then given a Pick status in order to generate a pick list Spots can be identified as a protein of interest using either the DeCyder Differential Analysis Software DIA or BVA module DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 109 ER Spot picking A 541 Identifying proteins for picking using the DIA module The DIA module is used to identify protein of interest for picking when performing small scale experiments utilizing two samples on a single gel e g control treated samples Assignment of POT is performed on all spot maps loaded into the DIA module Proteins assigned as protein of interest can then be further evaluated in the BVA module prior to assigning a Pick status to those proteins The simplest means t
161. he images spread evenly across the images As the images are slightly different it will be necessary to scroll the two images separately This can be done by clicking on the Properties icon and selecting the Image View tab Deselect the Link image views when scrolling option Scrolling and Auto Center Settings I Link image views when scrolling I Auto center selected spots 7 After landmarking click on the Match icon and select the Match Pending and Landmarked option in the window which now appears DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 213 ED 1 Ill Processing the Preparative Gel and Generating a Pick List 8 214 Click Match Match options C Match All Match Primary Match Pending and Landmarked Cancel Help When matching has completed the vector lines which appear will be very long due to the difference in size of the analytical and preparative gel These vector lines can be hidden by clicking on the properties icon and selecting the Image View tab Deselect the Match vectors when Match Table is displayed option Spot Features IV Signature Annotation E r ara as Match vectors when Match Table is displayed It is only necessary to determine whether the matches between the master image and the preparative image are accurate The only matches that need to be confirmed are those that match to proteins assigned with a pick status Therefore instead of confir
162. he match quality value in the Match Table can therefore be used to rapidly identify incorrect matches when reviewing inter gel matching In addition to quality score for each matched spot being displayed in the match table each set of matched spots a protein are also assigned a global match quality score displayed in the Protein table This protein match quality score represents the spot within the matched group that has the highest quality score This protein match quality value enables the identification of poorly matched spots thereby highlighting matched spots in the protein set that may contain outliers All the match quality values for all matched spots in a protein can be viewed simultaneously in the Appearance Table facilitating the rapid identification of the outlying data point There is also a quality score value designated for each spot map which is displayed in the Spot Map Table This value refers to the average quality score for all matched standard spots on each gel Hence gels that are poorly matched possess high match quality values High spot map match quality values are often due to poorly run gels that cannot be matched efficiently without land marking see section 4 6 1 Note The match quality score is based exclusively on the surface profile of the spots on the internal standard spot maps Therefore the quality score cannot be calculated for experimental designs that do not contain an internal standard DeCyder
163. he selected spot in 3 dimensions not displayed in Spot Map Table mode 180 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial Il Employing an internal standard to Analyze Protein Changes Oy A o Graph View Graphical representation of the protein abundance data for a single spot across the different images in the analysis set Note The graph view is only displayed in Protein Table and Appearance Table AT mode In Spot Map Table ST mode the graph view is substituted for Experimental Design view see section 4 5 2 All the tables in BVA can be ordered by clicking on the headers at the top of each column Fie Edit View Process Help D d sm ma 2 F eee 99 AAA ABN Graph View Master No 973 o o o e o ona Standardised Log Abundance b J 3 9e 010 Unconfirmed 7 1e 010 Unconfirmed J 116 009 Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed 3 D View Uncorfed Unconfirmed Spot No 940 Position 638 402 Spot No 798 Position 682 377 PriSpot Map Protein ID Protein AC Name Comment T Pick I PTM a Y J Confirm X Mw Da Protein of Interest gt Data Control Panel Focus Secondary D ven AUN DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 181 ED 1 Il Employing an internal standard to Analyze Protein Changes Other than the icons t
164. ies of spots contained within the entire workspace 3 5 Data analysis The DIA module does not require the user to perform any form of spot boundary editing After spot detection the user only has two post detection tasks to perform 1 Creation and execution of an exclusion filter to remove non proteinaceous spots 2 Confirmation of detected spots This is used for small scale experiments to confirm expression differences in two samples run on a single gel This approach was employed to remove user to user variation from software analysis In house studies have shown that different users generate statistically different results if spot editing is permitted The spot detection process DIA in DeCyder Differential Analysis Software has been demonstrated to be highly accurate in house studies have demonstrated 98 accuracy The accuracy achieved without editing is taken as an acceptable level and is more than outweighed by the removal of user to user variation and higher throughput from not editing DeCyder Differential Analysis User Manual 18 1173 16 Edition AA DIA Differential In gel Analysis Module A 351 Spot exclusion Assigning an area of interest The automated spot detection algorithm in DeCyder Differential Analysis Software DIA may detect artifacts due to gel heterogeneity at the edges of the images These spots can be removed by setting an area of interest to define the gel only Those spots outside of the area of int
165. ifferential Analysis Software evaluation into a pick list format Ettan LWS 1 0 can read e describe how the Protein Identification tool is used to import the protein identification results from Ettan LWS data back into DeCyder BVA 8 2 Ettan LWS with samples generated within Ettan DIGE system There are two alternative ways to enter a sample generated within Ettan DIGE system into Ettan LWS workflow e asa protein tube or e asa gel with a pick list and an image file The two alternatives offer different levels of sample tracking For a protein tube tracking is available all the way through Ettan LWS workflow For a gel the tracking starts at spot handling The workflows are also different see Fig 8 2 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA LWS Integration 8 E tube Ettan DIGE System gt gel experimental design i DeCyder Differential Analysis Software Typhoon Variable Typhoon Variable Mode Imager Mode Imager Image file and Image file and picking gel picking gel Pick list Pick list Emm E XML Toolbox Ettan Spot Handling Workstation Ettan MALDI ToF Pro Text boxes show Ettan LWS workflow Bak DeCyder XML Toolbox DeCyder BVA Fig 8 2 Overview of the workflow for a sample generated within Ettan DIGE sys tem entered as a tube or as a gel E DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 141 ED LWS Integration 8 3 Enter a protein
166. ignificant protein changes under different experimental conditions Additional in built functionality allows post matching activities to be performed e Molecular weight calculation e Isoelectric point calculation e Database linkage e Statistical analyses e Spot pick list generation Data can be saved then re opened in a BVA file format Pick lists can be exported as either a text file or XML file for use in Ettan Spot Picker or Ettan Spot Handling Workstation respectively Data can also be exported in an XML format for querying in DeCyder Differential Analysis Software XML toolbox or copying and pasting into applications such as Microsoft Word and Excel 4 1 2 Graphical user interface The BVA graphical user interface is similar to DIA It is divided into four equally sized inter linked views Selecting a spot in for example the Image View will display the same spot in the Graph View 3 D View and the Table View DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 51 ED BVA Biological Variation Analysis Module 52 3 D View Spot No 940 Positior 638 402 Spot No 798 Position 682 377 ST File Edt View Process Help DSB sma VER As e Image View displays two gel images e 3 D View a three dimensional representation of the images localized on the spot selected e Graph View graphical representation of data e Table View tabulated data Master No 973
167. ilar experiment to that found in tutorial 1 However this tutorial utilizes gels that have already undergone spot detection in the DIA and have been exported in an XML format The processes involved in configuring the BVA without the Batch Processor are demonstrated See chapter 11 Tutorial III The processes required to analyze a picking gel for subsequent spot picking digestion and mass spectrometry analysis are demonstrated See chapter 12 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 157 EDB Introduction 158 Tutorial IV The tutorial employs all three software interfaces to ascertain protein expression changes in an experiment containing replicate gels in order to generate a pick list for Ettan Spot Picker The entire workflow is co ordinated via the Batch Processor thereby providing a fully automated process See chapter 13 The tutorials described above introduce the concepts and functionality of DeCyder Differential Analysis Software It is therefore recommended that these tutorials are performed first to gain a preliminary understanding of the software However it is recommended that various elements of the tutorials are used when analyzing an experiment incorporating an internal standard A workflow describing the various steps involved in an entire experiment are described in detail in appendix A DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial Using DIA modul
168. ill be sufficient So just as with image type resolution needs to be matched to the purpose of the scan The Table below shows the size of an uncompressed 1 x 1 image in different types and resolutions Resolution dpi 2 bit Black and 8 bit grayscale 24 bit color Kb white Kb Kb 400x400 20 158 475 300x300 11 89 256 200x200 5 39 118 100x100 1 9 29 e Dynamic range Dynamic range is the ability of an imaging system to quantitatively detect very dim and very bright features within a single image and is related to bit depth It is a measurement of the number of bits used to represent each pixel in an image and hence determines the number of colors or shades of gray grayscale that can be represented in a digital image The dynamic range of a system is a function of the analogue to digital converter the purity of the illuminating light colored filters and any system noise It is measured on scale from 0 0 perfect white to 4 0 perfect black and the single number given for a particular imaging system tells how much of that range the unit can distinguish Variations in dynamic range of a system impact the quality of the digitized image more than simple resolution does High end imaging systems are more sensitive to the range of colors in the spectrum and can record minor differences between two almost identical colors DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Several
169. image in DeCyder Differential Analysis Software and create pick list Convert pick list with LWS Pick List tool in XML Toolbox to an Ettan LVVS software format Import the converted pick list to Ettan LWS software Image Analysis Spot Handling MALDI MS Create report Detected Proteins in Gel in Ettan LWS software Convert results with Protein Identification tool in XML Toolbox to a format that can be read by the BVA module Final evaluation in DeCyder Differential Analysis Software Fig 8 1 Ettan LWS DeCyder XML Tools in Ettan DIGE system workflow To enable full tracking of the CyDye DIGE Fluor dye treated samples some extra information has to be added in the Sample Definition component of the Ettan LWS software DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 139 ED LWS Integration 140 The XML toolbox contains two tools to perform data conversion e WS Pick List converts pick lists from a DeCyder Differential Analysis Software workspace to a format that Ettan LWS can import e Protein Identification converts results from Ettan LWS in xml format to a format that the BVA module can import This section of the manual aims to e describe the deviation from the normal 2D MS workflow as described in Ettan 2D MS and LWS Software Laboratory Guide for samples generated within Ettan DIGE system e describe how the LWS Pick List tool is used to convert exported workspace data from the DeCyder D
170. in the matching process are landmarked Matching is then performed 12 Spots that conform to certain criteria such as statistical significance and above a certain ratio change are assigned as proteins of interest 13 These spots are confirmed for eventual inclusion into a pick list The individual stages involved in the experiment are now described more fully 11 3 Experimental design Four replicate gels are loaded with bacterial lysates as indicated in the table below Gel number Cy2 Cy3 Cy5 Gel 1 Standard Control 1 Treated 1 Gel 2 Standard Treated 2 Control 2 Gel 3 Standard Control 3 Treated 3 Gel 4 Standard Treated 4 Control 4 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial Il Employing an internal standard to Analyze Protein Changes Oy Each gel contains a standard sample to normalize control and treated samples against The standard sample is derived from control and treated lysates which are pooled in equal concentration The set of three images generated from a single gel are sequentially processed using the DIA module in order to perform spot detection and spot quantitation against the internal standard The data from DIA analysis is exported as an XML file for further analysis in the BVA module to evaluate protein abundance differences 11 4 Spot detection and quantitation Spot detection and quantitation by normalizing against the internal standard is perform
171. increase in abundance above the threshold mode set from the secondary image compared to the primary image DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 135 XML Toolbox 136 No of decreased spots Number of spots showing a decrease in abundance above the threshold mode set from the secondary image compared to the primary image Threshold mode Value above or below which spots are classed as being differentially expressed 2SD value 2 standard deviation of the spot ratio distribution 95 of the spots lie within this ratio for normally distributed data Image data Volume Sum of the detected pixel values above background within a spot boundary Peak height Pixel value at the X Y position of the spot This represents the highest pixel value within the spot boundary Spot slope Gradient associated with the 3 dimensional attributes of a spot map pair Gel data Volume ratio Ratio of the normalized volumes of a pair of spots from a spot map pair A value of 2 0 represents a two fold increase while 2 0 represents a two fold decrease whilst a value of 1 00 represents an unchanged spot Spot comment User defined comment on selected spot Picked Indicates whether the protein has been assigned for picking Coordinates Values giving the position of the center of mass of a spot on the spot map Protein ID Spot identifier which after confirmation assignment will appear with the relevant spot in the t
172. ing DeCyder Differential Analysis Software cccccccooconccononencnnnnnnnnn 9 1 7 Structure of DeCyder Differential Analysis Software o 9 2 Computer requirements and installation 2 1 Computer requirements cccccccocnnnncnonoonnnnnnnononnnnnnnnnnnnnnnncnonononos 220 sta A AAA Mike TSK 2 2 1 Installation with a previous version present 2 2 2 DO NOVO INSTA ALON cess ar aire 3 DIA Differential In gel Analysis Module 3T OVERVIOW iia e Ween aes Ad 3 2 Creating and Opening workspaces ccccccnococononononononononononnnnnnonnnoss 3 3 Spot detection and quantitation cccccoocoocccnionoccnononononaninononon 3 3 1 MATA A A lee ote A OS 3 3 2 QUIM alias 34 MIEWINE Spot lala rostros E E ncohed sctaacesnsted indi 3 4 1 TA TAR PR O es EL ri SS IN A O 3 4 3 TDI MW a tia 3 4 4 FLISTOBIGIN VOW sieisen a el des ai aa aaee T aa a a iaaa iait 3707 Dataanalysis a a red 351 SPOL OXCIUSION eane it sai al adidas 3 5 2 SPOb CORHNMMATION ii tds 35 3 Protein ofAnteres Elda ata 3 5 4 PEM assigned eee as a de cats 3 6 Customizing display Colors cccccccnonononoooooononoconnononononononccnnons 3 7 Saving exporting and printing ccccccnnnnconononononoconononononnnnnnnononos DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 4 BVA Biological Variation Analysis Module Al OVERS ED Eee re ne AE A de edge neterea teeter aitty 51 4 1 1 FUNCION Msi A a SN SEG Er rel s 51 4 1 2 S
173. ing the internal standard effectively eliminates gel to gel variation allowing detection of small differences in protein levels to be achieved Using DeCyder Differential Analysis Software system variability is minimized enabling expression differences identified by 2 D DIGE to be confidently assigned to induced biological change 1 2 The DeCyder Differential Analysis Software User Manual This user manual is broadly divided into two main parts the reference manual Chapters 1 8 and the tutorials Chapters 9 13 It is recommended that new users first work through the tutorials in order to gain a rapid understanding of the software s capabilities The tutorials are step by step guides that take the user through the main applications of the software by employing real data A CD containing the necessary files is required to perform each of the tutorials The tutorials are designed to be worked through without prior knowledge of the reference component of the manual The reference manual provides a detailed technical account encompassing all aspects of the built in functionality of DeCyder Differential Analysis Software which can be used as a source of further information for experienced users DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 11 tg Introduction to DeCyder Differential Analysis Software 12 1 3 Measuring differential protein abundance using Ettan DIGE system To compare protein abundance in differen
174. ions on standardized protein abundance Appearance Table Region within a BVA workspace where users can track all information on a particular spot from all the gel images Area of Interest User defined region outside of which any detected protein spots are excluded from analysis Artifact Rejection Filtering out of non proteinaceous signal Auto Level The stage in the algorithm in which spots are matched Bandwidth The transmission capacity of a communications channel usually expressed in bits or bytes per second Batch Processor DeCyder Differential Analysis Software module capable of performing fully automated co detection quantification and matching of multiple spot maps BVA Biological Variance Analysis module of DeCyder Differential Analysis Software BVA Batch list The spreadsheet in the batch processor depicting which spot maps are to be matched DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 275 276 Centre of Volume CoV Central tendency of the spot volumes on a spot map Co detection Simultaneous detection of labelled protein spots from two in gel images Comment User defined text string that can be linked to specific protein or spot map DeCyder Differential Analysis Software XML XML format used to transfer data between the various DeCyder Differential Analysis Software modules DIA Differential In gel Analysis module of DeCyder Differential Analysis Software capable of the full
175. ith the highest match quality values see section 4 6 4 3 Confirm randomly selected auto level 1 e g 5 and auto level 2 i e 10 matches by scrolling down the table 4 Confirm all matches on every gel time consuming and not recommended Note Areas of incorrect matching can be identified rapidly by examining the match vectors displayed in the Image View The vectors should be orientated in a similar direction across the gel If an area of the gel does not follow this pattern e g the vectors are perpendicular or cross it is highly likely that mismatches are present in that area and should be looked at closely Deciding whether a match is accurate can be aided by viewing the selected spot and the surrounding cluster in the 3 D View as well as looking at the matched spots in the Image View If the match is correct confirm the match by clicking the Confirm match button in the data control panel If the match is incorrect click on the Break match button which now becomes Add Match Click the correct spot on the match image If the spot on the match image has also been wrongly matched break this match as before Select the corresponding spots on the master image and the match image and click on the button entitled Add Match If a matched spot on the primary image is not present on the master for any reason it can be added to the master by clicking the Add to Master button A comment can be appended to each match in the commen
176. king the icon stops rotation The 3 D View can be rotated manually by dragging the mouse over the 3 D image Adjustment of the 3 D View can be performed using the mouse Holding down both mouse buttons and dragging the mouse upwards and downwards results in zooming in and out the 3 D View respectively The position of the spot image within the 3 D View can be adjusted by holding down the right mouse button then dragging the mouse over the 3 D View Data associated with each of the highlighted spots in the 3 D View are displayed beneath the corresponding spot DeCyder Differential Analysis User Manual 18 1173 16 Edition AA DIA Differential In gel Analysis Module The orientation of the scaling of the 3 D View can be reset to the initial viewing settings by selecting the Reset button above in the 3 D View Spot number Spot reference number unique to a co detected spot on a set of images from the same gel Position Gel X Y co ordinates of the co detected spot Volume Spot pixel volume Peak Height Largest pixel value within the spot boundary expressed with background subtraction Area Number of pixels within the spot boundary Pick Position Gel X Y co ordinates of the spot pick location Properties Properties associated with 3 D View are defined in the 3 D View properties window Selecting View Properties or selecting the Properties icon then selecting the 3 D View tab displays the 3 D
177. l 18 1173 16 Edition AA DIA Differential In gel Analysis Module Workspace Properties Properties associated with the workspace are defined in the Workspace Properties window Selecting View Properties or selecting the Properties icon then selecting the Workspace tab displays the workspace properties window DeCyder DIA Properties 3D View Tableview Colors Workspace Spot Display Image View Primary Image File name Gel 04 Cy2 Standard gel Dyetag Cy2 X Secondary Image File name Gel 04 Cy3 Treated gel Dye tag Cy3 X Tertiary Image File name Gel 04 Cy5 Control gel Dye tag Cy5 v General Estimated no of spots 2500 Dye chemistry Included spots 1628 DIGE min Exclude Filter Applied Cancel The dye tag used and the dye chemistry used can be selected from the pull down menus The dye tag label is automatically selected if the information is included in the image file name The remaining information is derived from the data processing performed 3 3 Spot detection and quantitation 3 3 1 Detection There are three distinct spot detection algorithms which are able to simultaneously process a different number of images derived from a single gel e Single detection one image e Double detection two images e Triple detection three images Single detection is performed on images of post stained gels used for picking a case where there is a single image associated
178. l Analysis Software analysis workspaces for other applications The XML format is partly used to transfer data between the different modules of the DeCyder Differential Analysis Software but it is also used to make data available for post processing by other software packages such as database building packages The DeCyder Differential Analysis Software XML Toolbox is a toolbox shell housing different tools for extraction of the DeCyder Differential Analysis Software data from the different XML files produced within DeCyder Differential Analysis Software This enables users to create tools to convert their data into text files or html files potentially other data formats can be supported for conversion Two basic tools are supplied to create Tabbed text files and Web tables 7 2 Opening the XML Toolbox module Click on the Toolbox icon to open the XML Toolbox The XML Toolbox can extract data in two formats Tabbed text files and Web tables Tabbed text files The Tab Separated Tables tool is used to export DeCyder Differential Analysis Software XML data in a tabbed text format for further data processing in other software that can import this data e g Microsoft Excel or SpotFire The data in the resulting output table is configured such that the top row consists of an identifying line of text containing the name of the source XML file and the type of data extracted from the file The second row in the output table is a tab separated
179. l Analysis User Manual 18 1173 16 Edition AA Tutorial III Processing the Preparative Gel and Generating a Pick List 12 Select the Autodetect Picking references check box Click OK to begin spot detection on the images Process Gel Images Algorithm Selection Co Detection algorithm Version 5 00 01 Description Co detection algorithm Performs spot detection in one or more images based on features in the image contrast Estimated no of spots 2500 Autodetect Picking references V Cancel Help Before going any further it is important to become acquainted with the main tool bar Dea Create Workspace What s this Open Workspace Print Save Workspace z Properties ProcessGels All Views Pick Filter Table View Exclude Filter 3D View Areain3D Histogram View Rotate 3D Image View Zoom In Brightness C ontrast Zoom Out Fit to Window SSS DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 201 ED 1 Ill Processing the Preparative Gel and Generating a Pick List E The user interface is divided into four main windows the Image View the Histogram View the 3 D View and the Table View 202 Image EH Decyder DIA File Edit View Process Help DEA Primary SYPRO gel Secondary ex Gelo 2 DELAS Histogram selections Scatter parameter Threshold mode Threshold 1 25D o Spot statistics Decreased 0 10 0 Similar 2340 Increased o 10 0 Workspace infomation
180. l Cy3 to select the primary gel image Load Primary Gel Image Look in Tutorial misc Gel 01 Control Cy3 gel My Recent Gel 01 Control Cy5 gel Documents Gel 02 Control Cy3 gel tll cel 02 Treated Cy5 gel My Network File name Gel 01 Control Cy3 gel Places Files of type Gel image files tif gel 5 When the Load Secondary Gel Image window appears double click on the image Gel 01 Control Cy5 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 161 10 1 Using DIA module for preliminary investigation of protein changes E 1042 Spot detection 1 After the images have been loaded select Process Process Gel images Process Gel Images Exclude Filter Re Normalise Protein Filter Unassign all Protein of Interest Assign all Protein of Interest as Pick 2 Inthe process gel images dialog box which now appears enter the value 2500 for the estimated number of spots see help file for an explanation of this value Click OK to begin spot detection on the images Process Gel Images Algorithm Selection Co Detection algorithm Version 5 00 01 Description Co detection algorithm Performs spot detection in one or more images based on features in the image contrast Estimated no of spots 2500 Autodetect Picking references I Cancel Help 162 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial Using
181. l Variation Analysis Module 76 4 8 4 Independent and paired analyses The Student s T test One Way ANOVA and Two Way ANOVA can be either independent normal or paired individual Both types are used to test the hypothesis that a variable in this case protein abundance differs between groups or experimental conditions The paired test is specifically used when each data point in one group corresponds to a matching data point in the other group s A typical example would be the same group of patients before and after a treatment The unpaired tests are more general techniques that can be used to test whether standardized protein abundance differs between groups and does not require that the groups be paired in any way or even of equal sizes The following graphs are examples illustrating independent and paired tests Standardised Abundance Non diseased Diseased Experimental Group Independent test Abundance of a specific protein in five diseased individuals compared to a group of five non diseased individuals Data points are independent since there are different individuals in each group DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN Individual 1 Individual 2 18 1 6 1 4 1 2 Individual 3 Individual 4 t Individual 5 0 8 0 6 0 4 0 2 Standardised Abundance Pre treated Post
182. l value Sample ID User defined sample identification Comment User defined comment Match Quality Morphological similarity metric describing deviation of internal standard spot to an average internal standard spot in protein set yA DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 101 ED BVA Biological Variation Analysis Module E The columns displayed in the appearance table can be changed using 102 the Appearance Table Properties dialog box see next page DeCyder BYA Properties Image View 3D View Graph View Database Colors Spot Map Table Match Table Protein Table Appearance Table Table Column Order and Visibility Select columns to display and the column order by drag and drop Left Column No Status Image Gel No Type Label No of Spots Matched Function Group Group Description ndition1 Condition2 Sample ID Comment Match Quality K K K K K K K KIKI 7 j KK K v v Y M Right Column Restore to default order and selection Default Condition Labels Condition 1 Label Condition Condition 2 Label Condition2 Cancel Table Column Order and Visibility The selected column titles are displayed in the Table View Clicking and dragging the column titles in the list sets the order of the columns in the Table View Clicking Default restores the original settings 4 13 3 Spot annotation Displaying an
183. le Appearance Table Image View 3D View Graph View Database Colors 3D Settings Spot Margin for displayed spots IV Show caption colors Cancel Help 4 4 3 Table View The contents of the Table View is dependent on BVA mode selected Similarly the editable properties associated with the Table View are dependent upon the mode selected and are discussed in detail in later sections The data within the table can be sorted into ascending or descending order by clicking on the column headers of the table No Status Image Type Label No of Spots Matched Function Group Group Description Condition1 Condition2 Sample ID TY DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 57 ED BVA Biological Variation Analysis Module 58 4 5 Defining spot map attributes All spot map attributes are assigned in the Spot Map Table mode Select View Spot Map Table or select the toolbar icon to work in the Spot Map Table mode if a new BVA workspace has been created the interface will automatically open to the Spot Map Table This mode consists of an Image Table View and Experimental Design View 4 5 1 Function assignment Spot Maps can be assigned with up to four functions that indicate the role of the Spot Map in the BVA processing The Spot Map functions are Analysis A Spot data is included in quantitative analysis Those that should not be assigned as analysis images may include preparative gels for spot picking and ima
184. lection Default Condition Labels Condition 1 Label Dose Condition 2 Label Time Cancel Cw Help Table Column Order and Visibility The column titles selected will be displayed in the Table View Dragging the column titles in the list defines the order of the columns in the Table View Clicking Default restores the original settings Condition Labels Labels for condition 1 and condition 2 e g time dose can be entered see section 4 8 8 62 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN E 4 6 Inter gel matching All spot maps are matched to a master image sequentially The spot matching algorithm is a pattern recognition algorithm that matches one single spot in one gel to a single spot in another gel based on it s neighboring spots 4 6 1 Landmarking Landmarking allows the user to manually define matched protein spots in order to improve the accuracy of the gel to gel matching process In some cases images from different gels may vary sufficiently to require landmarks to be set Landmarking is performed in the Match Table mode Select View Match Table or select the toolbar icon to work in the Match Table mode This mode consists of an Image 3 D and Table View To use a spot as a landmark the spot must be present on both the match image and the master Click on the Magnify Image View icon to display the gel images only The EH i
185. list of column headers and the following rows consist of the actual tab separated raw data DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 129 XML Toolbox Tab Separated Tables Web Tables i 130 To utilize the Tab Separated Tables tool click on the Tab Separated Tables button Web tables The Web Tables tool is an example of how the DeCyder Differential Analysis Software XML format in combination with XSL and other web technology can be used to customize reports in HTML format The Web Tables tool can thus be used as a template for user designed report tools To utilize the Web Tables tool click on the Web Tables button The interface for both tools is similar 7 3 Extracting data Loading the XML files XML files can be loaded at the top of the tool page by either browsing for a file then clicking Open or by typing the file pathway directly into the list box then pressing enter on the keyboard The tool verifies whether the selected file is a valid XML file exported from DeCyder Differential Analysis Software DIA or DeCyder Differential Analysis Software BVA The user is alerted if the selected file is not valid Load XML file generated from DIA or BYA lc Firman Tutorial images v5 Temp Tutorial data filesyTutd File type DIA Type of proteins in the output table Once the DeCyder Differential Analysis Software XML file has been opened select the types of proteins to be included in the outp
186. lization procedure 244 Original image Image is partitioned into a two dimensional array of square sections Each division will be The scanning device then collects a single value to represent the entire square After the computer has received values for each section the entire image can be used to form a which it then reconstructed pixel transmits to the using the pixel computer x y values B 3 Formatting graphic files Once an image has been acquired it is converted to a particular file format for storage There are a number of widely used image formats such as TIFF GIF etc and knowing which format to use is a key issue Some formats are proprietary and only useful in the program used to modify or acquire the image Others are useful for exchange between programs and computer platforms or for presentation in web pages Image files include a certain amount of technical information which is stored in an area called the image header or tag The image header may be of use in displaying the image e g length and width in pixels identifying the image e g name or source or identifying the owner Scientific images should be acquired and archived using an information preserving or non destructive format The compression algorithms associated with some file formats actually distort some of the pixel information when the file is saved while others do not Loss of data
187. ll Views Properties Alt Enter 5 Click on any of the four views available to expand that view to fill the screen When you have finished click on View and select All Views from the menu above to return to the All views display Alternatively use the icons displayed on the main toolbar 6 If after detection has been completed the hour glass turns back to a cursor the Table View remains empty click on the Properties icon to bring up the Properties dialog box Alternatively click on View Properties By default this will open on the Workspace tab 7 Change to the Table View options by clicking on the Table View tab 8 By selecting and deselecting the check boxes the various categories of spots can be selectively displayed Deselecting all the check boxes results in the Table View being blank Similarly spots can be selectively displayed in the Image View using the Spot Display tab The Table View tab works on OR logic i e a spot only needs to conform to one criterion to be shown in the table Select the Similar Decreased and Increased check boxes as indicated on next page DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 223 Tutorial IV Fully automated identification of differentially expressed proteins _ _ lt lt gt gt gt gt A42 DeCyder DIA Properties 3D View Table View Colors Workspace Spot Display Image View Included spots Similar IV Decreased IV Increa
188. llowing selection of spot volume ratios within a finite limit Spots can also be selected on the basis of their physical characteristics i e area max volume max peak height to ensure that spot selection only occurs on spots that can potentially be successfully picked digested then analyzed by mass spectroscopy The location of the spots on the X and Y axes can also be filtered in order that spots near or on the edge of the gel are not present in the pick list or to limit the eventual pick list to high or low molecular weight proteins The various filters can be used simultaneously to select spots on multiple features This filtering can take place on the whole spot population or only those confirmed by selecting the Restrict to confirmed spots check box After entering the filter criteria select the Filter button to ascertain the number of spots that will be picked If the number of resultant spots is unsuitable the stringency of the filter can be adjusted to produce an optimal number of spots for selection and subsequent picking Click OK to accept the spot filter parameters If spot confirmation was not performed prior to the picking filter then the spots selected for picking can be visually inspected and confirmed The protein of interest status of spots can be removed by selecting Process Unassign all Protein of Interest The DIA file containing the protein of interest selection information can then be exported as an XML file File
189. llows on from Tutorials I and II The first two tutorials concentrate on finding differences between different biological groups This tutorial describes how to excise these spots for further protein identification analysis An experimental design is determined as described in tutorial II with the addition of a separate preparative gel containing two reference markers and stained with SYPRO Ruby The analytical gels are analyzed in DeCyder Differential Analysis Software to ascertain the proteins of interest as described in Tutorial I The preparative gel is analyzed in DeCyder Differential Analysis Software DIA The reference markers are assigned in DIA alternatively this can be done in BVA The preparative gel spot map is exported as an XML file and added to the BVA workspace that contains the analytical gels The preparative gel spot map is landmarked and matched to the master gel of the analytical set The protein of interest assignments are transferred from the analytical set to the preparative gel The proteins of interest are assigned a Pick status then the pick locations are visually inspected and edited if necessary A pick list is exported from the preparative gel and the gel with pick list is then taken to Ettan Spot Picker or Ettan Spot Handling Workstation for spot excision See the relevant User Manual for instructions 12 3 Experimental design Ettan DIGE system and DeCyder Differential Analysis Software are
190. lows the user to determine whether legends defining the color coding of groups will be displayed Y axis settings The Y axis setting options enable the user to change the Y axis scaling e Automatic allows optimization of the Y axis scaling e Manual allows the user to define the maximum and minimum values on the Y axis e Manual with Automatic increase allows the user to define the maximum and minimum values on the Y axis but adjusts the Y axis values if the data points fall outside the manual defined values 4 8 Protein statistics 4 8 1 Opening the protein statistics dialog box DeCyder Differential Analysis Software BVA possesses several statistical analysis methods that can be employed to ascertain whether changes in expression of specific proteins are significant between samples from different experimental groups To open the statistical analysis dialog select menu Process Protein Statistics Heb Match Unmatch Primary Protein Stati Calibrate pI and Mw Protein Filter Unpick All Unassign all Protein of Interest Assign all Protein of Interest as Pick Alternatively the statistical analysis dialog can be opened using the toolbar icon DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 73 ED BVA Biological Variation Analysis Module The dialog box contains various options allowing statistical analysis of protein data from spot maps loaded in the DeCyder Differential Analysi
191. lter icon to open the Protein Filter dialog box see section 4 9 for further information DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Spot picking ea Protein Filter Filter action IV Assign Proteins of Interest IT Assign Pick status General filter settings I Select all TT Restrict to confirmed proteins I Restrict to proteins present in gt spot maps Select proteins with IT Student s T test value T Average Ratio or IT Average Ratio IT Average Ratio I One way ANOVA value IT Two way ANOVA Dose value I Two way ANOVA Time value T Two way ANOVA Interaction value Properties for proteins in Master y spot map I Volume and lt TT X co ordinate and lt I Y co ordinate and lt Filter All protein spots detected can be assigned by selecting the Select All check box Alternatively proteins can be filtered for picking based on the statistical analysis results produced i e based on the statistical criteria described in section 4 8 This filtering can take place on the whole spot population or a subset of the proteins or spot maps Selecting the Restrict to confirmed proteins check box ensures that only proteins with a confirm status are filtered Alternatively selecting the Restrict to protein present in check box ensures that only spots that appear on the designated number of spot maps are filtered Filtering can be limited to spots of a
192. lue 9 Directory 1 16 Offsetto _ L_EnttyN Offset 11 next IFD or null 4 bytes of zero Value DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 245 246 e Gel Image File Format Images scanned on the Typhoon are stored as GEL files which are a variation of the 16 bit TIFF and are purely gray scale files with one bit for each pixel These i images have very wide dynamic ranges typically images with 10 or 1 100 000 levels of signal resolution are possible The GEL format uses a square root algorithm to compress the possible 100 000 levels of an image into the 65536 levels available this is located in the TIFF private tag domain The square root compression also provides higher signal resolution at the low end where small changes in signal are more critical This is important when discriminating between two small values The TIFF file will assign the same number to two values that differ by a small amount whereas the GEL file accurately represents the data The recommended instrument for scanning Ettan DIGE system gels for DeCyder Differential Analysis Software analysis is the Typhoon Variable Mode Imager B 4 Elements of the digital image e Pixel intensity value Each of the pixels that represent a stored image has a pixel intensity value which describes how bright that pixel is This intensity value represents a measured physical property i e emitted light This value is the average f
193. ly The picking location of all picks is reviewed to assess whether the pick location requires editing see section 5 6 Spot No 811 Pick Pos 735 394 735 394 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 215 ED 1 Ill Processing the Preparative Gel and Generating a Pick List 216 13 To edit a pick location zoom in on the selected spot in the image view Select Edit Edit Pick Location then place the hand shaped cursor that appears in the image view over the centre of the pick locations that require editing then drag the pick circle to the desired location Select Edit Edit Pick Location to exit this mode To return pick locations to their original position select Edit Restore Default Pick Locations 12 4 4 Exporting the Pick List Now that the SYPRO stained gel has been matched in and the pick proteins have been reviewed the Pick list has to be exported for use on the appropriate Ettan picking system Select File Export Picking List from and select the Pick Spot Map to export a pick list Type in an appropriate file name for the pick list The pick list can be saved as either a text file or an XML file by appending the appropriate file extension to the file name txt or xml The text and XML file are used for Ettan Spot Picker or Ettan Spot Handling Work station respectively Click OK The pick list file and the actual SYPRO Ruby stained gel are taken to the appropriate picking system The pick
194. mage on the left represents the master image in the Match Table mode the left gel image is always the master image while the image on the right represents one of the images to be matched to the master To commence setting landmarks click on the button entitled Landmark Landmakmede Mode in the data control panel at the bottom of the workspace this al button will now become yellow or to a color the user has assigned as the landmark color In the drop down menu in the Image View title bar select the image for landmarking If all the spots are unmatched the spot boundaries are red to indicate this If the spot boundaries are green in an unmatched set of spots then the images are from the same DIA file thus landmarks are not necessary DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 63 ED BVA Biological Variation Analysis Module Tf the spots on the images are not clearly visible it may be necessary to alter the contrast brightness settings of the images Click on the Contrast Brightness icon and alter the position of the bars to alter the contrast and brightness of the images until only the most intense spots are visible To set a landmark click on a clearly defined spot on the master image the spot boundary should become magenta color set as default Now select the spot on the match image which corresponds to the master image spot so that this spot also becomes magenta A few seconds later a vector line
195. mage view icon on the tool bar to have a full screen view of the gel images 3 Next click on the Properties icon to bring up the properties window 4 Select the Spot Display tab and deselect Similar Increased and Decreased so that the check boxes are identical to those shown in the figure below Click OK DeCyder DIA Properties 3D View TableView Colors Workspace Spot Display Image View Included spots M Similar Decreased Increased Excluded spots IV Picking references and pick locations FT Picked spots Cancel Apply Help 5 To set an area of interest select Edit Define area of interest Using the rectangular target pointer which now appears drag the mouse to draw a rectangle around the gel ensuring that edge artifacts are SY DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 165 ED Tutorial Using DIA module for preliminary investigation of protein changes removed It does not matter if the Reference Markers are inside or outside the Area of Interest F Decyder Dla Control Control dia File Edit View Process Help EEEIEE CEELEN T Pick T PIM a TT Protein of Interest I Exclude Focus Primary Image NUM 10 4 4 Gel artifact removal A 3 D View clearly shows if a detected spot is a gel artifact rather than a protein spot due to the very steep sides and pointed top of an artifact compared to the smooth curve of a protein spot 0 Actual Protein Dust Particle
196. ming matches in the match table they should be confirmed in the protein table Click on the PT icon Go to the Protein Table tab of the properties dialog box and select the Protein of Interest I only check box Click OK Protein Table Filter IV Protein of Interest I only I Proteins assigned as Pick only DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial III Processing the Preparative Gel and Generating a Pick List 12 E 11 Inthe table only proteins assigned as protein of interest will be present In the Image View have the master gel as the primary image and the preparative gel as the secondary image Start at the top of the table and click on the first spot check if this spot is matched to the master and if so whether it is matched correctly If this is the case select the Pick check box in the data control panel However if there is an incorrect match switch to the Match Table and correct the match Then move back to the protein table and look at the next spot in the list If the spot is not present on the preparative gel and hence cannot be picked move to the next protein in the table this protein will not have a Pick status Whilst reviewing the match accuracy of the preparative gel the pick locations should be verified simultaneously 12 Spots assigned with a Pick status have a yellow transparent cylinder and a yellow circle denoting the picking location in the 3 D and image views respective
197. n Protein Table mode This is accomplished by opening the Properties dialog window and ensuring that Spots present in table only check box in the Image View tab is selected and the Proteins assigned as Pick only check box is selected in the Protein Table tab DeCyder BVA Properties Spot Map Table Match Table Protein Table Appearance Table Image View DView GraphView Database Colors Include in Image View Spots present in table only Spot Features Y Signature IT Annotation I Match vectors when Match Table is displayed Scrolling and Auto Center Settings Link image views when scroling Auto center selected spots Picking Reference Data Picking references and pick locations Primary Image View Default radius of picking references 5 100 DeCyder BYA Properties Image View 3D View GraphView Database Colors Spot Map Table MatchTable Protein Table Protein Table Filter TT Protein of Interest 1 only IV Proteins assigned as Pick only Table Column Order and Visibility Select columns to display and the column order by drag and drop Left Column MPo WiMaster No Status Protein ID Protein AC Appearance W Paired T test Paired Av Ratio Vi1 RMANOVA M 2 RMANOVA Condition1 2 RMANOVA Condition2 M2 RMANOVA Interact MPicked Pick Spot Vol MFunction pl Mw ViName Comment Appearance Table Match Quality Default diameter of picker head 5 50 eo ET
198. n filter These are the spots that are of most interest and they can now be manually confirmed 10 6 2 Spot confirmation Three options are available when you start to confirm your spots 1 Only confirm spots that are Increased blue and or Decreased red in their abundance This is the recommended option Select View Properties and go to the Table View tab Ensure that the Decreased and Increased options are selected and that the Excluded box is not selected Also ensure that Confirmed is selected unconfirmed is not Click OK DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 171 10 1 Using DIA module for preliminary investigation of protein changes Only confirm spots that have been assigned with a protein of interest status recommended option when the protein of interest assignment option has been performed Select View Properties and go to the Table View tab Ensure that only the Picked spots checkbox is selected 3 All spots Decreased red Increased blue and Similar green are manually verified This takes approximately 1 5 hours for 1000 spots this is not recommended as only those spots that are increasing or decreasing in abundance are of interest Select View Properties and go to the Table View tab Ensure that the Similar Decreased and Increased options are selected and that the Excluded box is not selected Click OK When confirming spots a decision is made whether the spots selected
199. n in Table View is also updated automatically Threshold Indicates the volume ratio selected in the threshold mode pull down menu e g the volume ratio represented by 2 model S D 2 S D Indicates the volume ratio for 2 S D based on the raw data In a normally distributed data set 95 of data points fall within this value Spot statistics Displays information on the spot population illustrated in the histogram view based on the user defined threshold settings e Decreased the number of spots classified as decreased in their abundance in the primary image compared to the secondary image DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 41 DIA Differential In gel Analysis Module 42 e Similar the number of spots classified as not differentially expressed in the primary image compared to the secondary image e Increased the number of spots classified as increased in their abundance in the primary image compared to the secondary image The Table View Image View and Histogram View can be adjusted to selectively display any of these subgroups by selecting the Spot Display and Table View tabs in the properties dialog box then selecting de selecting the appropriate check boxes The number of spots designated as excluded and included designated either manually or by the Exclude Filter are displayed see next section for more details Workspace information Displays frequency data on the various categor
200. n internal standard to Analyze Protein Changes 11 1 Objectives Tutorial I describes the analysis of a simple experimental design using only the DIA module to evaluate samples where an internal standard is not possible due to sample scarcity This tutorial extends to a more complex experimental design incorporating an internal standard with several replicate samples This tutorial describes how to find proteins that exhibit statistically significant changes between control and treated groups of bacterial cultures using DeCyder Differential Analysis Software The procedure in this tutorial can be applied to any two groups of protein mixtures with either replicate gels or replicate biological samples The tutorial outlines the various stages in experimental design sample organization protein detection and quantitation gel matching and statistical analysis 11 2 Overview The following describes the various stages involved in identifying all proteins that are differentially expressed in a given system and which therefore warrant further investigation The stages are 1 An experimental design is devised that will generate statistically significant results a design which minimizes or eliminates in gel and gel to gel system variation 2 Eight sample lysates that form the basis of the experiment are prepared This consists of four lysates derived from four bacterial cultures treated with benzoic acid and four control flasks that have not be
201. n internal standard to Analyze Protein Changes ON on the right represents one of the images to be matched to the master 3 To begin setting landmarks click on the button entitled Landmark mode at the bottom of the screen this button will now become yellow From the Select Match Gel scroll down menu circled in the following image select the first image All the spots should appear red to indicate that they are unmatched If you select an image and the spots appear green both images are from the same DIA image analysis and thus landmarks are not necessary therefore select another image 4 Ifthe spots on the images are not clearly visible it may be necessary to alter the contrast brightness settings of the images Click on the contrast brightness icon and alter the position of the bars to alter the contrast and brightness of the images until only the most intense spots are visible 5 To set a landmark click on a clearly defined spot on the master image the spot boundary should become magenta Now select the spot on the match image which corresponds to the master image spot so that this spot becomes magenta a few seconds later a vector line should appear showing that the spots have been matched and the landmark has been set 6 Repeat this procedure until approximately 10 landmarks have been set It is recommended that landmarks are evenly distributed across the image as this aids the matching process After landmarks have De
202. n the images must be entered before clicking OK For example a mammalian lysate run on an 18 cm pH 4 7 Immobiline DryStrip and a large format gel such as the Ettan DALT electrophoresis unit 20 cm x 26 cm a value of 2500 for Estimated Number of Spots should be satisfactory If all the spots have not been identified the spot detection process can be repeated with a higher number of estimated spots It is recommended that this value be overestimated to compensate for the detection of non proteinaceous spots on the image e g dust particles which are subsequently excluded from the analysis DeCyder Differential Analysis User Manual 18 1173 16 Edition AA DIA Differential In gel Analysis Module A 3 3 2 Quantitation Numerical data for individual spots are calculated e g volume area peak height and slope Spot volumes sum of pixel intensities within the spot boundary are always expressed with background subtracted Background is subtracted on a spot specific basis by excluding the lowest 10 percentile pixel value on the spot boundary from all other pixel values within the spot boundary The spot volume is the summation of these corrected values Spot ratios are calculated volume of secondary image spot volume of primary image spot This ratio indicates the change in spot volume between the two images These ratio values are normalized so that the modal peak of volume ratios is zero since the majority of proteins are
203. ncnonnnnns 165 10 4 4 Gel artifact TeMoval oooooooninocinnccoconnonncnnonnnonannn conan nono nenonos 166 10 4 5 Ascertaining the 2 S D standard deviation threshold values coastal ti dk as iban is iaa 168 10 5 Analysis of control treated gel occccccoccccccccoocccncnononananonononnn 168 10 5 1 Selecting gel IMages ccccccocoononcnncoooonnnonnocannnnconnonnnnnnonnnnn 168 10 52 Spot detection ienirt A ees 168 105 3 AA ea aa ian R D A 168 TOS Setting Bthresnle Paisvveiee Nevehe venta Me a 169 10 6 Assigning protein of interest ooocccncocccncconononcnanoconnonannnononona 170 10 6 1 Selecting spots for picking ccccoonoconccooccaccononanancconncannnnos 170 1O 6 2 SPOUCONTMIMAUON eee ea ba las ieee 171 10 6 3 Exporting the Pick List and physically excising spots HOM the pel mesrine aana E A aA EAEE agre aA 172 Tutorial II Employing an internal standard to Analyze Protein Changes TET Objectives mitra ti 173 TEZ OVEIEW pid onena A a e nesi en ner ae ANA 173 11 3 Experimental design ccococococcccnncoocnnonocooonanonncononnnnononononnnnncnonos 174 11 4 Spot detection and quantitation oooooooccccccccocaccccnonannccnononos 175 114 1 Selecting gel IMages cocccocooonocnnccononanonnccannnanonnnonananonnnnn 175 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 11 42 Spot detection and quantitation ss ssasa11a101n0aa1ra1ra1n0a 176 1143 Viewing the DIA worksp
204. nd subsequent volume ratios when using an internal standard are recalculated automatically The merging process can be reversed by selecting Edit Split Previously Merged Splitting merged spots results in all the associated spots being unmatched DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 69 ED BVA Biological Variation Analysis Module A 46 6 Match Table The Match Table displays spot specific information Pos Match Table row number Master No Protein spot number on Master Spot Map Status Match confirmation status confirmed or unconfirmed Master Protein spot co ordinates on Master Spot Map Match Protein spot co ordinates on Match Spot Map Type Types of match Unmatched Unmatched spots Auto Levell Spots matched after first level of matching Auto Level2 Spots matched after second level of matching Manual Spots matched by the user Added Spots that have been added to the master Comment User defined comment regarding matched protein spots 4 6 7 Match Table properties Properties associated with Match Table are defined in the Match Table Properties dialog box Selecting View Properties or selecting the Properties icon then selecting the Match Table tab displays the Match Table Properties dialog box DeCyder BVA Properties Image View 3D View Graph View Database Colors Spot Map Table Match Table Protein Table Appearance Table
205. nd TrEMBL Full text search any word Swiss 2D page Search by AC Swiss 3D image database Search by AC PROSITE Search by AC Compute pl Mw ID or AC search Restore Defaults Add coa To add a database to the list click on the Add button SSS DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 95 ED BVA Biological Variation Analysis Module 96 Add new web database label and address Address A placeholder string has to be included in the address and will be replaced by the protein s ID or AC Eg http www expasy ch cgi bin get sprot entry BVA_PROTID BVA_PROTIDH Protein ID BVA_PROTACH Protein AC BVA_PROTIDACH Both Protein ID and AC Test Select a Protein ID or AC that will T g ie replace the placeholder string Cancel The name of the database is entered in the Label text box The web address URL in the format specified is entered in the address text box The address should contain a placeholder string which will be replaced by the protein s ID or Accession Number AC depending on the string is used and the information available There are three different placeholder strings e BVA_PROTID The database will be available if there is information in the Protein ID column of the Protein Table e BVA_PROTACH The database will be available if there is information in the Protein AC column of the Protein Table e HBVA_PROTIDACH The database will be available if ther
206. ndling methods e In Ettan MALDI Control Methods add MALDI methods DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 145 ED LWS Integration 85 XML toolbox LWS Pick List The LWS Pick List tool is used to transform DeCyder Differential Analysis Software data into an Ettan LWS 1 0 format The tool takes a DeCyder xml file exported from BVA or DIA extracts the spots in the file that are selected for picking in DeCyder Differential Analysis Software and creates a new xml file for import to Ettan LWS software 146 8 5 1 Preparations before XML conversion 1 If possible base pick lists on information in a BVA workspace rather than a DIA workspace It is a lot easier to find relevant protein spots to pick in the BVA module as it includes an overview of all experimental conditions In BVA workspace use a workspace including spot maps from a picking gel that contains real pick references A spot map from the gel to be used for picking needs to be assigned as a pick spot map before exporting the XML workspace file In DIA workspace use a workspace based on gel images from a picking gel that contains two real pick reference markers If a DIA workspace is used to generate a pick list for LWS it will not be possible to import protein data for that pick list into the BVA module using the protein identification tool Assign the pick references in DeCyder Differential Analysis Software Export the workspace from BV
207. ne 58 Pi kine reference crac idad Taer A decd ore ERE 56 108 Pixel Post translational modification oooooccconnninccccnnnniccccnnccncnnno 48 94 Preparative gel arnis inpia er anr eler herrer 107 Primary Mage anuncia 29 A ON 49 Process multiple IMAGES ccccccoccccccncoconnnononooonnnononononanonononos 226 PROS Eran 95 ProteinvAGs a E E sieate 98 Protein confirmati0N ea eaea esaa aae e ERE 94 Protein data cis ld 151 Protein Fiter O 90 112 Protein lD aca ais dci la al a het aa a 98 148 POEM Names A tar aa 148 Pr teiotintereSt srren cameos tees ascites eae 94 Protein lable ta Ls tock vets A a av ero done ches coh 98 PIM le a tae kal toa aR ame edo 48 94 Q QUAIIV SCONG 3 ss Ang eR Re aed de 67 Quantitation 2 he ete ecg ante 33 53 R Reagents tiie Seale Si aren ei ki Ss i LASER 272 Recommended Workflow ooocconocccnoocncononcncnoncnononononanannononoos 237 Flow Diagram sh ani dnd With Shaves 238 Flow DiagraM 2 SE En En sieni iaa 240 Flow Diagram rat os dl ate ROS wake 241 Reference Manual cescccssescecesseceesseeeesseeeceeseessusetesssatesesstens 11 Reference markers mia 108 Related documentation ccoccoccnoncncnononnnaonnnononcnononononanannnnonos 273 Related Proc sell o DICES 271 Repeated Measures ccccccccccncnnnnnnnnonnnononnononnonononnonanancncncininninos 83 RESOLU tata ler lt tds 247 Run bateh processor ss arie rarere eas tad 235 S Sample Attribute Templates ccccoocccccccnncocccnccon
208. nfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Uecoaficmad Similar Similar Decreased Similar Decreased Similar Similar Similar Similar Similar Similar Similar Decreased Similar Similar Protein ID DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Comment T PIM TT Exclude I Pick TT Protein of Interest Confirm Focus Secondary 3D View NUM 163 10 1 Using DIA module for preliminary investigation of protein changes 164 The four views are all linked Clicking on a spot on the Image View highlights the spot in magenta in the Histogram View The spot is also represented three dimensionally and is highlighted in gray in the Table View 3 To display the entire gel image click on the Fit to window icon 4 After detection it is important to save the workspace Select File Save Workspace As and place an appropriate name in the dialog box which now appears The detected spots are of three types increased decreased and similar colored blue red and green respectively in the Histogram View 5 If after detection the hourglass turns back to a cursor the Table View remains empty click on the Properties icon to bring up the properties dialog box By default this will open on the Workspace tab 6 Change to the table options by clicking on the Table View tab 7 By selecting and deselecting the
209. ng within Ettan DIGE system A scececasche ens E T 144 yA DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 7 3 9 XMLtoolbox LWSPiclelist steeds detaches 146 8 5 1 Preparations before XML conversion ooococcccccooccooooadoncnnnnnnss 146 8 5 2 Using the LWS Pick List tool ooconinincncoonooconnnnanannananonnnnos 147 8 5 3 After XML CONVeYSION ooocococcaniccocacanracarin rara ranra rra canario rn nn 148 8 6 XML Toolbox Protein Identification ooonnnnnccnnnnnncccnnnccccccs 148 8 6 1 Preparations in Ettan LWS before XML conversion 149 8 6 2 Using the Protein Identification 1001 150 8 7 Importing protein data templates into the BVA module 151 8 8 Database queries in DeCyder BVA occcnoccccncccccococononococonanonononos 151 Tutorials Introduction 9 1 DeCyder Differential Analysis Software Structure ccccccnnnnn 155 92 SCOperottutona Sa dl eed 157 10 Tutorial Using DIA module for preliminary 11 investigation of protein changes TOO UV id AA ai it 159 LORO dae ad 159 10 3 Experimental design ococococoncccnoccocnnonocononnnonncononnnnnncnonnncnnnnonos 160 10 4 Analysis of control control gel ccocccoccncccnooccccncconononnnnnnnnnnns 160 10 4 1 Selecting gel Mages ccccooconccnnonononananonooonnnononnonanananononnnn 160 104 2 Spot detec Mia a A AE 162 10 4 3 Assigning an area Of INterest oooooooococnccoooanccnooonannn
210. not up or down regulated This ratio parameter is referred to as the volume ratio In all DeCyder Differential Analysis Software DIA tables the volume ratio is expressed in the range of 1 to for increases in spot volumes and 1 to oo for decreases in spot volumes Values between 1 and 1 are not represented hence a two fold increase and decrease is represented by 2 and 2 respectively not 2 and 0 5 When using either double or triple detection algorithms the images displayed in the primary and secondary views can be selected using the drop down in the Image View title bar Spot quantitation is automatically recalculated upon alteration of the primary and secondary image When using single detection the volume ratio value is left blank since there is no secondary image Note The volume ratio is displayed in the Table View in this manner but all data analyses are based on the log of the normalized ratio measurement see Appendix C for further details DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 33 DIA Differential In gel Analysis Module 34 3 4 Viewing spot data 3 4 1 Image View Image View simultaneously displays both primary and secondary gel images Five toolbar functions are associated with the Image View All these functions can also be accessed through menu pull down options indicated in parentheses View Image View Expands the Image View to fit the workspace View Zoom Zoom In Z
211. notation Various protein data elements can be displayed on the Image View The annotation can be defined using the Image View Properties dialog box See Section 4 4 1 for details DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN Positioning the annotations The position of the annotations in the Image View can be altered to ensure that the different annotations are clearly visible This feature is useful when one annotation obscures another annotation To alter the position of an annotation select Edit Move Annotations so that a tick appears next to this option The annotation labels in the Image View can then be dragged to the new positions 4 13 4 Customizing display colors The colors used in the various views can be customized by selecting the Colors tab in the Properties dialog box DeCyder BYA Properties Spot Map Table Match Table Protein Table Appearance Table Image View 3D View GraphView Database Colors Match Colors Unmatched Auto Level 1 matched Auto Level 2 matched Manually matched Added to Master Match vector Landmark Spot Colors Base color Selected Pick assigned SOO 1999990 Picking references and pick locations Annotation Colors Annotation background Annotation link Spot Map Colors Master Spot Map Primary Spot Map Secondary Spot Map Restore to default colors Match Colors The spot bo
212. nsure the Autodetect Picking references check box is selected then click OK The reference markers will then be detected automatically during the spot detection process Process Gel Images Algorithm Selection Version 5 00 01 Description Co detection algorithm Performs spot detection in one or more images based on features in the image contrast Estimated no of spots 2500 Autodetect Picking references IV Cancel Help Alternatively the reference markers can be assigned manually Click on the Magnify Gel View icon to show the gel images only and select Edit Define Picking Reference Using the circular mouse cursor that appears in the Image View click on the center of the left reference DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Spot picking ea marker of the primary image Zoom into the area of this reference marker by holding down the left mouse button to draw a rectangular area around the marker Clicking on the Properties icon and selecting the Image View tab can alter the size of the target The value entered in the Default radius of picking references can be set so that the circle in the Image View is sufficiently large enough to accurately surround the reference marker The position of the circle can be altered by selecting Edit Edit picking References Place the hand shaped cursor that now appears on the circle then drag the circle over the center of the reference marker Move the target so t
213. nt protein spots and allows match confirmation The match table consists of an Image View 3 D View and Table View Each of the views are linked in such a way that selecting a spot in the Image View will display that spot in 3 dimensions and highlights that spot in the table The matches in the Table View are of two types Auto Level 1 high probability matches and Auto Level 2 lower probability matches By default the two types of matches are displayed differently in the Image View Auto level 1 and Auto level 2 spots are represented by a darker and lighter spot boundary respectively The boundaries of the unmatched spots are colored red these colors can be altered by clicking on the properties icon and selecting the colors tab The Colors window allows the user to define colors to the different matches DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial Il Employing an internal standard to Analyze Protein Changes Oy a _ Qz gt DeCyder BVA Properties Spot Map Table Match Table Protein Table Appearance Table Image View 3DView GraphView Database Colors Match Colors Unmatched Auto Level 1 matched o Auto Level 2 matched o Manually matched Added to Master e Match vector Landmark Spot Colors Base color o Selected Pick assigned o Picking references and pick locations Annotation Colors Annotation background gt Annotation link 9 Spot Map C
214. o assign POI status is to manually choose spots then select the Protein of Interest check box Pick I PTM 7 Confirm I Protein of Interest Exclude a The spots can also be selected using various criteria via the Protein Filter Click on the Protein Filter icon to open the Protein Filter dialog box Protein Filter Filter action IV Assign Protein of Interest I Assign Pick status General filter settings Pick all Restrict to confirmed spots Select spots with M Volume Ratio or Volume Ratio I Volume Ratio Area I Max Volume I Max Peak Height TX co ordinate TY co ordinate Filter 110 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Spot picking ER In the Protein Filter select the Assign Protein of Interest check box and deselect the Assign Pick Status check box In this way the proteins that meet the filter criteria will be given a Protein of Interest status All spots detected can be assigned with protein of interest status by selecting the Pick All check box Alternatively spots can be filtered on specific spot properties Protein spots exhibiting a change in expression are commonly analyzed Therefore selecting proteins of interest on the basis of volume ratio is useful for such spots Spots can be selected for decreases or increases in order to select spots that have diverged notably from each other Alternatively spots can be selected for decreases and increases a
215. o assign protein spots that are on 75 of spot maps i e restricted to proteins present in gt 9 spot maps with a T test score less than 0 01 and with an average ratio greater than or equal to 1 5 or less than or equal to 1 5 and a pick gel volume of 1x10 1x108 the pick gel setting the parameters to those identical to the figure below Click OK Protein Filter Filter action IV Assign proteins of Interest General filter settings Select all Restrict to proteins present in Select proteins with Student s T test value IV Average Ratio or IV Average Ratio I One way ANOVA value I Two way ANOVA Condition value gt o Two way ANOVA Condition2 value gt 0 Two way ANOVA Interaction value gt Properties for proteins in Master Gel w IV Volume and lt 1649 I X co ordinate and lt IT Y co ordinate and lt Cancel DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial IV Fully automated identification of differentially expressed proteins A 1355 Saving the Pick List Immediately after setting the protein filter parameters the Set Pick List File Name dialog box automatically appears 1 Browse to select the folder in which to save the pick list for convenience it may be placed in the folder Tutorial IV 2 Enter an appropriate name of the pick list file Using the Save as type pull down menu the file extension for the pick list can either be a text or XML
216. o move between the different screens the main Create Workspace Open Workspace Save Workspace Spot Map Table Match T able Protein Table Appearance Table Exclude Filter Protein Statistics Calibrate pl and Mw Protein Filter Areain3D Rotate 3D 182 BVA tool bar is identical to the DIA tool bar OSGi sim em ar Sr Be 9 28 VMAa yaa NN 11 5 2 Function assignment What s this Print Properties Display All Views Magnify Table View Magnify 3D View Magnify Exp Design View Magnify Image View Contrast and Brightness Fit to window Zoom Out Zoom In 1 Ensure that the Spot Map table icon is selected This screen consists of an Image Experimental Design and Table View Select View Table E View or click on the Magnify Table View icon to display the Table View only The Table View shows the images that have been loaded into the workspace and the number of spots that have been detected on them Note A set of images from the same gel will have the same number of spots since the DIA detection algorithm is designed to detect the same number of spots on images from the same gel DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial Il Employing an internal standard to Analyze Protein Changes Oy Ly File Edit View Process Help D amp G stor T sp E 2 239 EN A 34058 e Spot Map Table Status Image Gel No
217. ocated under the File menu 1 Export Result Table Exports all the data in the Table View in a text format that can be opened in a spreadsheet 2 Export pick list Exports the proteins which have been assigned for picking by the user in the form of a txt or xml file that can be recognized by Ettan Spot Picker or Ettan Spot Handling Workstation 3 Export Spot Maps Exports all the data from the DIA workspace in an XML format that contains all the data generated in the DIA workspace This can be opened by either the DeCyder Differential Analysis Software BVA or the DeCyder Differential Analysis Software XML toolbox DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN 4 BVA Biological Variation Analysis Module 4 1 Overview 4 1 1 Functionality DeCyder Differential Analysis Software BVA processes multiple Ettan DIGE system gel images performing gel to gel matching of spots allowing quantitative comparisons of protein expression across multiple gels BVA processes gel images that have undergone spot detection in DIA The BVA module utilizes the XML files generated in DIA Section 3 7 together with the original scanned image files The images are then matched to a single master image identifying common protein spots across the gels Various experimental designs can be assigned in BVA facilitating the statistical analysis tools to highlight proteins that demonstrate s
218. ogical Variation Analysis Module EN Printing Select File Print to open the Print dialog box Print r Image View IV Primary Image IV Secondary Image M Graph View IT Both side by side Table View IT Spot Map Table Number of ry copies 7 E IV Match Table IV 3D View E I Protein Table I Appearance Table AA Printer Name APBAMEOSSHP LaserJet 4050 Series PC w Properties Status Ready Type HP LaserJet 4050 Series PCL 6 Where LPT1 Comment Preview Cancel Help Multiple check boxes can be selected for printing the required workspace views DeCyder Differential Analysis Software supports the following printers e HP LaserJet 4000N e HP color LaserJet 5M e HP 2500C e HP DeskJet 895 Cxi e HP DeskJet 950C e HP Laser Jet 4100 DTN SSS DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 105 ED BVA Biological Variation Analysis Module 106 Using the clipboard Any aspect of the different views within the workspace can be copied to the clipboard for pasting into another application Depending on the BVA mode different views will be available for copying Click on the part of the workspace chosen for pasting then select Edit Copy the part of the workspace selected is described in this pull down menu The clipboard can then be pasted into other applications Exporting data Data associated with the entire workspace can be exported in an XML based format DeCyder Differential Analysis Software XML by Sel
219. olors Master Spot Map a Primary Spot Map o Secondary Spot Map Restore to default colors Cancel The vector lines in the Image View indicate the positional difference for the same protein spot on different gels The accuracy of the matching process can now be checked within the match table 2 Order the table by clicking on the column header Type so that all the Auto Level 1 matches are at the top of the table 3 To confirm a match in the Match Table View click on the first spot in the table ensure that it is an actual protein spot The spot should be selected in the Image View in magenta Zoom into the area of the selected spot by holding down the left mouse click and drawing a square around the selected area 4 Decide whether the spot in the match image corresponds to the spot on the Master image Deciding whether a match is accurate can be aided by viewing the selected spot and the surrounding cluster in the 3 D View as well as looking at the matched spots in the Image ee DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 187 ED II Employing an internal standard to Analyze Protein Changes 188 View If the match is correct change the Match gel image using the Select Match Gel scroll down menu to test the matching accuracy on the other gel images 5 Ifthe match is incorrect click on the Break Match button at the bottom of the screen which now becomes Add Match Click the correct spot on the match image
220. omatically applied later in the experiment to remove all non protein spots from all the gel images The Batch Processor is run to perform a fully automated comparative analysis of the control and treated samples This analysis involves the following steps Running a series of four sequential DIA analyses for each of the four gels e Performing spot detection and calculation of the spot properties e Removing non protein spots using the previously ascertained exclusion filter parameters e Normalization of the protein spots using the in gel linked internal standard e Loading of the derived spot maps and associated data into the BVA module e Inter gel matching of spot maps e T test analysis of proteins in control and treated samples e Generation of a pick list for Ettan Spot Picker 13 3 Experimental design In order to generate statistically valid data a minimum of three replicates must be used These can be either biological replicates from each group or if no biological replicates are available then replicate gels must be employed For biologically relevant answers to a hypothesis it is strongly advised that biological replicates of each group are used The greater the number of biological replicates the more biological variation is taken into consideration and therefore the more biologically relevant the results are to the system being investigated For the purpose of this tutorial we describe the analysis of four re
221. omatically by normalizing spot volumes against the internal standard Fig 1 3 The co detection algorithm ensures that the internal standard and the quantified analytical spot have an identical spot boundary This results in a highly accurate and robust protein quantitation DeCyder Differential Analysis Software utilizes experimental design incorporating an internal standard and performs gel to gel matching on the standard samples DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 17 ED Introduction to DeCyder Differential Analysis Software 18 A Differential In gel Analysis DIA In gel spot matching and determination of ratios standard sample Pooled std Gel 1 1 05 Gel 2 1 2 Gel 3 11 yo o o e Sample 1 g Sample 3 Sample 5 11 E El 124 12 Ae ee o Pooled std Pooled std o e e Sample 2 Sample 4 Sample 6 B Biological Variation Analysis BVA Quantitative comparison of protein abundance across multiple gels Standard Log Abundance _ Sample 3 and 6 _ 7 increased abundance Samples 2 4 and 5 abundance unchanged Sample 1 decreased abundance T T Standard samples Fig 1 3 A DeCyder Differential Analysis Software utilizes an internal standard to a aid spot matching between samples within the same gel and b generate a ratio of protein abundance between the proteins
222. on Single Detection one image C Double Detection two images C Triple Detection three images Cancel 4 Browse to locate the folder entitled Tutorial III in the Tutorial data files folder Select the SYPRO gel image then click Open to load the DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 199 ED Tutorial Ill Processing the Preparative Gel and Generating a Pick List E Load Primary Gel Image Look in O Tutorial Iv x ens isc B Gel 01 Cy2 Standard gel My Recent Gel 01 Cy3 Control gel Documents Gel 01 CyS Treated gel Ei Gel 02 Cy2 Standard gel Gel 02 Cy3 Treated gel Desktop Gel 02 Cy5 Control gel Gel 03 Cy2 Standard gel Gel 03 Cy3 Control gel Gel 03 Cy5 Treated gel Gel 04 Cy2 Standard gel Gel 04 Cy3 Treated gel Gel 04 Cy5 Control gel My Computer Pick gel File name Fick gel Files of type Gel image files tf gel gt Cancel 5 As the image was stained with the SYPRO Ruby stain the initial o te image contrast may require adjusting Select the contrast brightness icon and alter the bars to make the image clearer gt Primary x ar 12 3 2 Spot detection 1 After the image has been loaded select Process Process Gel Images process E Pr Gel Im Exclude Filter 2 In the Process Gel Images dialog box which now appears enter the value 2500 for the estimated number of spots see section 3 3 1 200 DeCyder Differentia
223. on AA 79 ED BVA Biological Variation Analysis Module _ _ lt lt gt gt gt gt A42 Protein Statistics Type of statistical tests Independent tests normal Paired tests use sample ID Statistical tests IV Average Ratio IV Student s T test Population 1 Population 2 Dption s Dption s 4 5 7 A 3 Members 3 Members FT One Way ANOVA between different groups FT Two Way ANOVA between Dose and Time Cancel Help The T test p values and average ratios will be displayed in the protein table 3 The spots in the Protein Table can then be sorted by clicking on the T test column header This will order the table so that the protein spots exhibiting the most significant changes are listed at the top of the table Since the T test is two tailed the statistical significance of both increases and decreases in protein abundance quantified are ordered together A positive average ratio value indicates an increase from population 2 to population 1 the order is stipulated when defining groups in the protein statistics dialog box Conversely a decrease in abundance is denoted by a negative average ratio 80 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN Protein Table T test and Av Ratio 6 3 Pos Master No Protein ID Protein AC Mame Appearance T test Av Ratio 1 ANOVA 2
224. on label can then be entered into the appropriate text boxes DeCyder BVA Properties Image View 3D View GraphView Database Colors Spot Map Table Match Table Protein Table Appearance Table r Table Column Order and Visibility Select columns to display and the column order by drag and drop Left Column No Status Image W Gel No Type Label No of Spots Matched Function Group Group Description Dose Time Sample ID Z Comment Right Column Restore to defaylL ordeLand selection Default Condition Labels Condition 1 Label Dose Condition 2 Label Time Cancel DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 87 ED BVA Biological Variation Analysis Module The experimental conditions of each gel image can then be entered at the bottom of the spot map view Conditions can be entered as integers to represent the exact conditions e g dose 1 2 3 n can represent a series of 5 mg increments in a drug treatment Up to two conditions can be designated for each spot map Group Control Description Condition Condition2 LE o Color Add Edit Name Remove Note It can therefore be seen that when assigning a two condition experimental design that the Condition labels must be defined for a Two Way ANOVA test When assigning a single condition experiment the data is assigned into Groups for One Way A
225. on the analytical gels select the Protein Filter Dialog icon The Protein Filter selects protein spots based on user defined criteria such as Student s T test value average ratio or volume The protein filter is therefore used as a filter to find proteins that are interesting which can then be confirmed 2 Ensure that the Assign Proteins of Interest check box is selected and that the Assign Pick status is not selected This results in the proteins successfully filtered being given a Protein of Interest status For the purpose of this tutorial assign for selection protein spots with a T test score less than 0 01 and with an average ratio greater than or equal to 1 5 or less than or equal to 1 5 and a gel volume between 10 and 10 Click on Filter and it will tell you how many spots meet the criteria These values can be adjusted and the filter button clicked again to see how many spots now meet the criteria The purpose of this filtering is to narrow down the users search for interesting proteins that change significantly Click OK DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 193 ED 1 II Employing an internal standard to Analyze Protein Changes Protein Filter Filter action IV Assign Proteins of Interest Assign Pick status General filter settings Select all I Restrict to Confirmed proteins Restrict to proteins present in spot maps Select proteins with IV Student s T test value 0 01
226. on to bring up the Properties dialog box By default this will open on the Workspace tab Change to the table options by clicking on the Table View tab By selecting and deselecting the check boxes the various categories of spots can be selectively displayed Deselecting all the check boxes results in the Table View being blank Similarly spots can be selectively displayed in the image view using the Spot Display tab The Table View tab works on OR logic i e a spot only needs to conform to one criterion to be shown in the table DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial III Processing the Preparative Gel and Generating a Pick List 12 DeCyder DIA Properties Workspace Spot Display Image View 3D View Table View Colors Included spots IV Similar IV Decreased IV Increased IV Excluded spots IV Unconfirmed spots IV Confirmed spots IV Picked spots Cancel Selecting different protein spots on the images shows that some of the spots that have been detected are in fact either dust particles or artifacts from the gel To remove these artifacts an Exclude Filter must be used on the images 12 3 3 Assigning an area of interest Due to gel heterogeneity at the edges of the images there are artifacts that need to be removed These can be removed by setting an area of interest This function only works with the filter If an area of interest was set before detection all the
227. onancnoconinananass 142 Sample Definition A a a a 145 HODES a O wees 143 SAVES E A T A E T 49 104 E 282 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA o Scaler pala meten tada eh ces 40 Scierra WS ici idad 143 145 A A taming dar serende nat 142 144 Secondary IMAGE na a ate 36 A NO 55 A aden aa a ee ete 39 Sporared rer xs Aena Ae E O 37 SPOUCONTIKMATON tasado ls ales 46 Spot detection 7 i rta iio talados 31 single double triples cesos aceso dat stk de 29 SPOL EXCIUSION csi Seer a rt tira 43 Spot M prattrib ute ia ta daa 58 Spot Map function eto a Wav a lia h 58 Spot Map Table MOd sesaria licita cian tenses 58 Spot Merging ceiien aai ELTRA rer vre se dear dees 68 Spot number ooo ce eeceeeccccccessssceceecessseececcesssseeeeceessseeeceesstsaeeess 37 SPOEPOSILION ita ee ian eae 37 SBOE ral aorta tasas cote 33 Spot Statisti s dai ae od ITA 41 SPOLVOIUMME A tats rial 33 37 Standard report Detected proteins in gel 149 A 73 Students test AL eb reas 75 78 SWISS PFO ti da td 95 97 T able VIEW stre sagaer taka tr cee ie rai en denn ress 28 38 parameters sc did A A A ge 39 Tagged Image File Format TIFF oooooncnnncinocccicnconnnconnnnonncnos 245 template Spot Map Conviasa ln lesa ici heii iaa older 58 ME O tates ls 41 Trademarks dei a it 3 TEEMBL a o cie aid arden Peas Za dao ce aah a 95 TS a ll to dde 11 Two Way ANOVA ococcococccccoonnccooncncoononconnnoconnnnnnonnnno 75 85 98 Two
228. one item in each list is selected the resulting output contains four different result tables Multiple selections are possible in all lists The content of the four data lists also changes depending on whether an XML file is exported from the DIA or BVA module e Tabbed text files Select a category of DeCyder Differential Analysis Software data to include in the result output from the pull down menu Depending on the selection of category and type of XML file selected a number of items are displayed in the list box below For the Experimental data and Protein data categories multiple selections are possible in the list of data items but for the Image data and Gel data categories only a single selection is possible Extracting Data Once the data output has been specified the data can be extracted by clicking the Extract data button The data extraction process can take from a few seconds to several minutes depending on the size of the selected file the amount of data extracted and the performance of the computer Whilst the data is being DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 131 XML Toolbox 132 extracted a progress window displays the size of the XML file Once extracted the Result Table is displayed in a separate browser window A floating Save As button is present in the upper right hand corner of the output window This button can be used to save the extracted output directly to a designated file
229. ononancnonononononononos 208 12 4 1 Preparing the WOrkSPACt ccoooocccncoooooccconoooonnnoconononnnaconiconos 208 12 4 2 Editing picking reference Markers scenerne 211 1243 Matching and landmarking ccmccococncccnonooooccccononananaconinonos 212 T244 Exporting the PICK LIST eis ceed diana tr iia tasa 216 13 Tutorial IV Fully automated identification of differentially expressed proteins 13 1 ODjectiVe manni aoee sys Salted elaine bei ase eh e 217 132 OE ica 217 13 3 Experimental desir ae Seaweeds 218 13 4 Protein spot filtering Optional oooonnnnnnninnnnnnnnnnonnoiacnnrcnns 219 134 1 Selecting gel images iii dei acid 220 T842 SPOt detection eas a e ia eden i Gtk 221 13 4 3 GOL anida OVA as 224 13 5 Processing multiple images ccooooocccccnnooconnncnononnnnnocnnononanonononos 226 13 5 1 Setting up the batch proCesSOf cococooconccnccoconnconocoaaannononanon 226 13 5 2 Assignment of spot map attributes 2 229 ERAN EA A ea ane a ai kk fe AA a 231 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 9 SRA 1 A a n Innere 232 13 55 Savin the Pick Sicilia ita iaa ii 233 13 5 6 Saving the batch workspace ooocccccccooooncccocooaananococonananonons 234 13 5 7 Running the batch proCesSOF cccmccocoocccnnooononoconocananonononnon 235 Appendices Appendix A Recommended workflow for analysis of multiple gels 237 Appendix B Understanding the digital image 243 Appendix C Spot processing algorithms
230. ooms in to selected region of the image View Zoom Zoom Out Zooms out of selected region of the image View Zoom Fit to window Fits the entire gel image to Image View window View Contrast Brightness Changes the brightness and contrast of the Image View Raising the positions of the slide bar increases either the contrast or brightness Selecting the Apply to all Images check box results in linking the controls to all images Altering the contrast and brightness does not change the raw pixel data contained within the image hence subsequent analyses are not effected Zooming in and out of the image can also be performed using the mouse by dragging the mouse over a square area of the image to be zoomed Dragging the mouse from top left to bottom right results in zooming into the image whilst dragging the mouse from bottom right to top left result in resizing the image to fit DeCyder Differential Analysis User Manual 18 1173 16 Edition AA DIA Differential In gel Analysis Module Properties Properties associated with Image View are defined in the Image View properties window Selecting View Properties or selecting the Properties icon then selecting the Image View tab displays the Image View properties window DeCyder DIA Properties 3D View TableWiew Colors Workspace Spot Display Image View Default radius of picking references 20 Default picking head diameter 20 v Auto center selected spots
231. ooooocococancncnnncnononnnnncococonananananano no nonnnnnns 76 Installation srair O 23 Isoelectric point calculation cccccccccnnncnnnnononononanonanononnnnnnnos 91 K Keyboard shortcuts cooooccococcccnoocnonononccnononononcncnononoconnnncnoncncnnns 267 L kandmarking erario 63 boad XML files ras dais E E SEEE 130 M Manual Spot exClUsiON dineti diia 46 Master image iia di eee 58 63 Mateh contitmatiO totor tl toda 65 Match Table ao 70 A O A A 63 65 Max peak height cascade 38 Max vol Mee dd ARNE ENES SEE SAS RRS FR S NEDRE cal 38 Model CUIVES siese p teks fro ind ives be Arn aye erased ees 40 Molecular weight calculation oooooooccncccccccconoooncnnnonononananoninnnos 91 Multiple Maestra oii set eens he 226 MW NalUG ida dd 148 N NCBI rac techie Ren ee ds ae lcd ld reba cd ce 151 NOISE aida did A eed eal 249 NOM ProteInSPOUS sti ici 44 Normalization A tenia ele hice Boy ata rn ASLE 254 DMA e ee ede 251 0 One Way ANOVA onccccocccccconccononcncconnnnononononnncnn nnnnos 75 83 98 Open Works pater cocinas eyed tna teas 30 Original DeCyder BVA workspace occcococcnncccccoconcnnncnonnnass 148 151 P Palred analyses ii inthe tees lee ect 76 O 257 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 281 ad datar nar a 76 258 Patents andikicenices gt ii ds 3 Peak electa li ad rita ll 37 Alva o dd eds ol eta ao LAS 149 AAA IRA O A dette soca 110 PiEkKINSiIMage a ei cria e pe
232. or or from DeCyder Differential Analysis Software BVA module The resultant spot map table is illustrated 2 Post treated DIGE Min A DIGE Min Cy2 M A Standard DIGE Min Cy3 2572 2572 A 2 Post treated 2 DIGE Min Cys 2572 2572 A 1 Pre treated 2 DIGE Min Cy2 2507 1901 A Standard DIGE Min Cy3 2507 1901 A 1 Pre treated 3 DIGE Min Cys 2507 1901 A 2 Post treated 3 DIGE Min Cy2 2353 1943 A Standard DIGE Min Cy3 2353 1948 A 2 Post treated 4 DIGE Min Cy5 2353 1948 A a Pre treated 4 DIGE Min Cy2 2298 1705 A Standard DIGE Min Cy3 2298 1705 A 1 Pre treated 5 DIGE Min Cy5 2298 1705 A 2 Post treated 5 The experiment assesses the difference in protein expression between two experimental groups of paired data therefore a paired Student s T test is applicable The Student s T test and the Paired Tests check boxes must therefore be selected in the statistics dialog box The Average Ratio check box can also be selected to calculate the magnitude of any protein expression change between groups DeCyder Differential Analysis User Manual 18 1173 16 Edition AA E Example proteins Graph Yiew Master No 580 o 1 04 1 02 E 3 0 98 3 E 0 96 a 0 94 3 i 7 Protein Table T test and Av Ratio Post treated Pre treated Pos Master No Status Protein 1D Protein ac Appearance Paired T testa 1108 549 Confirmed 20 20 A M 6 8e 005 1109 552 Unconfirmed 20 20 A M 0 039 1110 553 Unconfi
233. or the whole area covered by the pixel A pixel s address is denoted by its row and column co ordinates in the two dimensional image e Pixel or bit depth Pixel or bit depth referred to as bit depth from now on refers to the amount of information allocated to each pixel in a graphic image Pixels have different bit depths which determine how many grayscales are available to the image Most image file formats store grayscale information in the header section of the file An 8 bit image 2 or 256 grayscale values a 16 bit image 2 16 or 65 536 grayscale values Due to variations in how graphics programs and conversion algorithms work it is best to start off with as much bit depth as possible Large bit depth also increases the dynamic range available when collecting images of for example gels with protein or nucleic acid bands that are going to be quantified by pixel intensity of the bands When working with images that have high bit depths remember that to actually see this information on the computer the display must also be set to a high bit depth thousands or millions of colors For black and white or binary images pixels need only two bits of information black or white and hence the bit depth is 4 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Another type of image is the grayscale image which is one in which the only colors are shades of gray The number of grayscales used can vary but 16 bit systems h
234. ota aia RIAs 74 4 8 3 Overview of Statistical tests ooooonioonnnniiinnnnnnniccccccininancinonn 75 4 8 4 Independent and Paired analyses ooocccncocicuoaoooaononanannnnnnnoss 76 4 8 5 Student s T test amp Average ratio cccccmiminononnononnonanannnnnnnnnos 78 ALBO ANOVA 2 eco 81 AB One Way ANOVA netsen ited indies alae ae 83 4 8 8 TWO Way ANOVA ici tl El dd adios 85 4 8 9 Further statistical analyS8sS cccccooconoconononanononannnnnnnnnnnnnnnos 90 AsO Protein Eller o a lindas 90 4 10 Molecular weight Mw and isoelectric point pl 91 4 10 1 Entering Mw and pl of KNOWN proteins 2 91 4 11 User defined protein labelling ooooococcnonocococcnononccnonononanos 94 ART CONO tad dd 94 A NaNe ea eaten tot a e SR ental A 94 ATS COMME ies bind 94 ATLA Protein ore iii di dai 94 AAT A a enes EA en Uae i A be a orate ered oe 94 4 12 Database linking ica 94 AIPA Adding database Snan id di Po 95 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA o 4 13 Viewing protein data assenaar A a a 98 24 33 15 A Ricavtandoddedts 98 413 2 Appearance able iii dt iY 101 4 133 SpOt annotatiOD occoococnncconoccccnonenooncnnnnnonnonocononanononononcnnnns 102 4 13 4 Customizing display CO OFS cccococcccccnooooccccnonananancconnannn 103 4 13 5 Saving exporting and printing aserne 104 5 Spot picking A tah e aac iat ena as 107 5 2 Spot detection on the preparative gel
235. oup 1 and 2 may have the same condition 1 value both temperature 1 but different condition 2 value drug treated or control Groups 3 and 4 will have the other condition 2 value both temperature 2 with different condition 2 values drug treated or control DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN e Experiment 1 there is no variation in abundance from temperature A to B or from treated and non treated samples e Experiment 2 temperature B results in increased protein expression whereas drug treatment has no effect e Experiment 3 drug treatment results in increased protein expression whereas temperature changes have no effect e Experiment 4 both drug treatment and increasing temperature result in increased protein expression i e the two conditions are independent and P43 is not significant e Experiment 5 drug treatment only in conjunction with change in temperature results in an increased protein abundance i e the two conditions are synergistic hence P lt 0 001 P4 Po and P43 are labelled as 2 ANOVA condition 1 2 ANOVA condition 2 and 2 ANOVA interact in the Protein Table Defining conditions In order to perform Two Way ANOVA analysis the experimental conditions for each gel image must first be assigned Prior to this the condition labels can be defined by selecting the properties icon and selecting the Spot Map Table tab the conditi
236. our bacterial cultures treated with benzoic acid and four control flasks that have not been treated Since the gel to gel variation in this system is low gel replicates are not compulsory if biological replicates are available 3 Aliquots from each sample are taken and pooled to prepare a standard sample All three sample types standard control and treated are labelled with an appropriate CyDye DIGE Fluor minimal dye Cy2 Cy3 or Cy5 dye as described in the table overleaf Each sample is then applied to the gels 4 The samples are applied to the four replicate Ettan DIGE system gels and the gels are then run Three images are produced from each gel by scanning at appropriate wavelengths for the three fluors The recommended scanning instrument for this is the Typhoon Variable Mode 9000 series Imager DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 217 Tutorial IV Fully automated identification of differentially expressed proteins 218 Two representative images from a single gel are initially processed using DeCyder Differential Analysis Software DIA Differential In gel Analysis module The DIA module performs spot detection using all images loaded generating spot information for all images simultaneously in this case standard control and treated images The data is examined to identify appropriate values for a number of variables that are set in the Exclude filter or artifact filter These values are aut
237. plicate gels loaded with bacterial lysates as indicated in the following table DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial IV Fully automated identification of differentially expressed proteins Each gel contains a standard sample to allow normalization of all control and treated samples The Standard sample is obtained by mixing aliquots of control and treated lysates pooled in equal concentration Gel Number Cy2 Cy3 Cy5 SYPRO Ruby Gel 1 Standard Control 1 Treated 1 Gel 2 Standard Treated 2 Control 2 Gel 3 Standard Control 3 Treated 3 Gel 4 Standard Treated 4 Control 4 Gel 5 Control Treated The data analysis is performed in two parts using the various DeCyder Differential Analysis Software modules e Determining the protein spot filtering parameters e Fully automated multi gel processing using the Batch Processor module 13 4 Protein spot filtering optional In order to identify and exclude artifacts from subsequent analyses the DIA module is initially run on a representative pair of images from the same gel This is performed using the DeCyder Differential Analysis Software DIA Differential In gel Analysis module which identifies protein spots and allows subsequent filtering to remove dust particles and other gel artifacts The user can examine the results and specify a series of parameters that will automatically filter all spots that fail to meet the
238. ply Help In the Graph View the dashed lines link data points from sample spots to their respective standard data point Therefore in this experiment there are four matched control spots and four matched treated spots associated with four standard data points The standards are the same on each gel and hence the normalization calculates how samples change with respect to their in gel standard A ratio of sample to standard of for example 2 5 means that the sample protein is 2 5 times greater than its standard protein or a ratio of 2 5 to 1 When this methodology is used all standards are 1 Thus the standard is displayed graphically as 1 and the samples displayed relative to their standard The log of 1 is zero and therefore when the log standardized abundance is displayed all standards are zero DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial Il Employing an internal standard to Analyze Protein Changes Oy The protein table shows average ratio i e Average abundance of control Average abundance of treated as well as the statistical significance of the differences 11 9 Assigning spots as proteins of interest 11 9 1 Selecting proteins of interest This process can be performed prior to spot confirmation or after spot confirmation In some ways doing this first reduces the amount of time spent on spot confirmation yA 1 To select those proteins that have been found to be statistically different
239. pot which may be present in several Spot Maps AT Appearance Table The Appearance table is used to display data associated with a selected protein across the gels 4 2 Protein quantitation When using DeCyder Differential Analysis Software BVA for inter gel analysis it is highly recommended that an internal standard is used in the experimental design see Section 1 4 As discussed in the previous section the DIA quantifies the spots as a function of the internal standard This value is used in the BVA for analyses facilitating direct comparison of spot maps and is referred to as the standardized abundance The log of this value referred to as standardized log abundance is used to aid scaling in graphical representations and is employed in all statistical analyses If an internal standard is not used in the experimental approach DeCyder Differential Analysis Software can express spot volumes of specific proteins across gels as a ratio relative to the lowest volume spot of that protein This parameter is referred to as the abundance in BVA This latter approach is not recommended since inter gel quantitative comparisons are not as accurate as the quantitation using an internal standard It is not possible to perform the BVA statistical functionality with this non internal standardized data As with the standardized abundance log y of this value is often used to aid scaling in graphical representations referred to as log abundance
240. pression in two E coli bacterial strains incubated at 37 C over a 90 minute time period Experimental design Bacterial cultures derived from each strain were incubated at 37 C Cell sample aliquots were taken immediately prior to incubation then 30 60 and 90 minutes after commencing incubation The cell samples were lysed labelled with CyDye DIGE Fluor minimal dye then equivalent amounts of protein were subjected to 2 D gel electrophoresis as described in the table on the following page standards on individual gel not shown Triplicate gels were run and analyzed to account for experimental variation Each gel contained a CyDye DIGE Fluor Cy2 minimal dye labelled internal pooled standard with CyDye DIGE Fluor Cy3 and CyS minimal dye labelled test samples Assigning groups and conditions There are two conditions present in this experiment e Condition 1 two bacterial strains represented by 1 and 2 e Condition 2 Four sampling time points Pre incubation 30 60 and 90 minutes incubations are represented by 1 2 3 and 4 respectively DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 261 E The table below shows the designation of groups conditions and individuals for the spot maps generated from the 12 experimental gels The Cy2 dye labelled internal standard samples are omitted from the table below Item
241. r Excluded spots are not imported to BVA Volume Ratio Normalized Volume Ratio between co detected spots in the primary and secondary images Picked Pick designation indicates that a spot has been selected for picking see section 5 4 1 Max Slope Largest gradient associated with co detected spots Area Number of pixels within the spot boundary Max Peak Height Largest pixel value associated with co detected spots Max Volume Volume of the largest of the co detected spots Protein ID User defined protein identification manually entered in the Protein ID text box at the bottom of the screen Comment User defined comment manually entered in the Comment text box at the bottom of the screen Function Indicates whether a spot has been selected as a protein of interest denoted by the letter I in the column PTM Indicates whether a spot has been selected as a protein that carries a post translational modification denoted by the letters PTM in the column 38 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA DIA Differential In gel Analysis Module E AA The following diagram illustrates some of the parameters in the Table View associated with a spot slope Peak height area volume DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 39 DIA Differential In gel Analysis Module 344 Histogram View The histogram view is onl
242. r a single spot across the different images in the analysis set All the tables in BVA can be ordered by clicking on the headers at the top of each column DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial III Processing the Preparative Gel and Generating a Pick List 12 a Other than the icons to move from one mode to another the main tool bar is very similar to that seen in DIA OSE seaferar Ss 2 4 22 999 300508 98 Create Workspace J L What s This help prompt Open Workspace Print Save Workspace va Properties Spot Map Table Display All Views Match Table Magnify Table View Protein Table Magnify 3D View Appearance Table Magnify histogram View Match Dialog Magnify Gel View Protein Statistics Dialog Contrast Brightness Calibrate pI and MW Dialog Fit to Window Pick Filter Dialog Area in 3D Zoom Out Rotate 3D Zoom in 4 Ensure that the Spot Map Table icon is selected This screen consists of an Image and a Table View 5 Select View Table View or click on the Magnify Table View icon to display the Table View only The table shows the images that have been loaded into the workspace and the number of spots that have been detected on them Spot Map Table No of Spots Matched Function Group Description Condition Condition2 Sample ID 2 Matched Gel 04
243. r the found values Click OK DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial III Processing the Preparative Gel and Generating a Pick List 12 Exclude Filter Spot properties TT Filter confirmed spots I Slope gt IV Area lt 50 lt I Peak Height IV Volume lt 20000 Current Area of Interest Whole Image Cancel Help An example of a non stringent set of filter parameters that can be used to run a light filter is as follows Area 50 Volume 20000 5 Select the All views icon to display all four views 6 Export the data from the DIA file as an XML file for gel to gel matching in BVA by selecting File Export Spot maps 7 Save the exported file in the folder entitled Tutorial III in the Tutorial data files folder At this point the DIA workspace can be closed without saving the workspace DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 207 ED 1 Ill Processing the Preparative Gel and Generating a Pick List 208 12 4 Matching to analytical gels The SYPRO Ruby stained picking gel has to be matched to analytical gels in order to identify the proteins on the picking gel that show differential expression and hence require picking 12 4 1 Preparing the workspace 1 Double click the DeCyder Differential Analysis Software BVA icon on your desktop to open the BVA workspace 2 Select File Open workspace and browse to locate the BVA fil
244. ratios which fall outside this threshold in a control treated experiment have some level of significance As a general rule the higher the threshold set the more confidence that differences above the value is significantly different above experimental variation Histogram selections Scatter parameter Max Volur Threshold mode Manual v Threshold 1 45805 25 D 2 32553 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 169 10 1 Using DIA module for preliminary investigation of protein changes 170 Pp 10 6 Assigning protein of interest 10 6 1 Selecting spots for picking 1 To select those proteins that have been found to be different on the control treated gel select the Protein Filter Dialog icon This process can be performed prior to or after spot confirmation The amount of time spent on spot confirmation can be reduced by performing filtering first The Protein Filter selects protein spots based on user defined criteria such as volume ratio above a maximum volume or peak height The protein filter is therefore used as a filter to find proteins that are interesting which can then be confirmed Open the Protein Filter dialog window Ensure that the Assign Protein of Interest check box is selected and that the Assign Pick status is not selected This results in the proteins successfully filtered being given a Protein of Interest status For the purpose of this tutorial select to assign pro
245. rd of the protein spot circled in blue suggests that this protein is present in a greater abundance in sample 4 Reference to the internal standard indicates that system variation has resulted in this protein having an increased volume in gel 2 relative to gel 1 Hence the abundance of this protein is unchanged in samples 1 2 and 4 and decreased in sample 3 A further benefit of using an internal standard is that matching between gels is more straightforward The internal standard image is common between all gels in an experiment therefore matching can be performed between internal standard images which have the similar spot patterns Conventional 2 D electrophoresis requires matching between different samples on different gels which introduces differences in spot patterns from sample to sample and gel to gel variation Matching between internal standards allows matching between identical samples so variations in spot patterns are due only to electrophoretic differences The internal standard approach can be applied to two color or three color experiments by including the internal standard plus one sample or two samples respectively on each gel DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 15 tg Introduction to DeCyder Differential Analysis Software 16 To maximize the quality of the data obtained when working with Ettan DIGE system the correct experimental design should be implemented Table 1 1 shows an example o
246. resentation of DeCyder Differential Analysis Software DIA workflow BVA DeCyder Differential Analysis Software BVA utilizes the DIA exported XML files together with the original gel images to match protein spots on different gels Data is then subjected to statistical analyses to accurately assess protein expression changes occurring in biological replicates comparing different conditions treatments This multi gel approach which allows analysis of replicates provides greater statistical validity to findings The final data can be saved as a BVA file from which spot pick lists and XML files can be exported In addition data can be copied directly from BVA and pasted into applications such as Microsoft Word and Excel Pick list for a multi gel Image XML file experiment GEL TXT XML 1 Image XML file GEL XML file Data extraction using TA XML Toolbox Image XML file 1 GEL h BVA file U GEL Image XML file Fig 9 2 Schematic representation of DeCyder Differential Analysis Software BVA workflow DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorials Introduction 9 Batch processor The Batch Processor integrates both the DIA and BVA modules enabling fully automated processing of multiple gels with minimal user intervention The Batch Processor can be configured
247. rimental group assignment To generate statistical data for the proteins which differ between the control and treated groups it is necessary to identify the different experimental groups within the software The three groups involved in this experiment are Control Treated and Standard Group assignment is performed via the Experimental Design view 1 Select View Experimental Design view or select the Magnify Experimental Fa Design View icon 2 The standard images will automatically be assigned to the group Standard by the software if the word standard or std appears in the image name The standard images will therefore be located in the DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 183 ED 1 Il Employing an internal standard to Analyze Protein Changes folder entitled standard By default non standard spot maps are assigned as Unassigned To assign the control and treated images to their respective groups these groups must first be created To create a control group select Add button then enter Control in the Group text box and click Confirm The new group folder Control is subsequently displayed The treated group is similarly generated Spot maps are assigned to either the control or treated group folders by selecting the Unassigned folder then dragging and dropping spot maps from the center panel of the Experimental Design view to the appropriate folders on the left of the screen FR DeCyder BVA File Edi
248. rkspaces Spiking experi Browse BVA Filename c Firman Manual workspaces Spiking expe Set XML Dir c Firman Manual workspaces Spiking experi Browse Pick Filename C Fitman Manual workspaces Spiking exp Set DIA Batch Li Primary abe Fiter Gel01 Standard Cy2 Cy2 S0A0POVO Gel02 Standard Cy2 c Cy2 Dis Belch A S 0A0P 0V 0 Cy2 S 0A0P 0V 0 2c Cy2 S 0A 0P 0V 0 gt S 0A 0P 0V 0 Gel06 Standard Cy2 EVA Batchi List 5 040 P 0V 0 Gel07 Standard Cy2 c Cy2 S 0A 0P 0V 0 Gel08 Standard Cy2 c Cy2 S 0A 0P 0V 0 Gel09 Standard Cy2 c Cy2 S 0A 0P 0V 0 Gell0 Standard Cy2 c Cy2 S 0A 0P 0V 0 Gell1 Standard Cy2 c Cy2 S 04 0P 0V 0 Gel12 Standard Cy2 c Cy2 S 04 0P 0V 0 128 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA XML Toolbox 7 XML Toolbox 7 1 Overview Large amounts of data are generated when a workspace is created and analyzed in DeCyder Differential Analysis Software It is useful to be able to save this data in a format that can be efficiently stored and queried Data is exported from the different DeCyder Differential Analysis Software modules using a common XML format called DeCyder Differential Analysis Software XML XML is a structured universal tagged language with tags that can be custom designed making it easy to access data from DeCyder Differentia
249. rmed 20 20 A M 0 27 1111 554 Unconfirmed 20 20 A M 0 00044 1112 555 Unconfirmed 20 20 A M 0 011 1113 571 Unconfirmed 20 20 A M 738 005 1114 574 Unconfirmed 20 20 A M 0 33 1115 575 Unconfirmed 20 20 A M 0 011 huy 581 Unconfirmed 20 20 A M 0 55 1118 582 Unconfirmed 20 20 A M 0 00023 1119 583 Unconfirmed 20 20 A M 0 10 1120 585 Unconfirmed 20 20 A M 0 45 1121 586 Unconfirmed 20 20 A M 0 085 1122 587 Unconfirmed 20 20 A M 0 00043 588 Unconfirmed 20 20 A M 0 20 y Ms ess IE ef Fig D 1 Statistical data displayed for protein 580 The independent T test p value indicates that there is no significant difference in the mean expression of protein 580 from pre to post drug treatment groups However when data pairing is accounted for the Paired T test p value indicates that there is a significant change Protein 580 expression levels appear to consistently increase regardless of the initial protein abundance SY DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 259 260 Graph View Master No 1142 Post treated 12 A Pre treated aie gi i 3195 Sl 095 3 E os 5 085 08 3 3 3 3 5 2 kA E E Protein Table T test and Av Ratio Post treated Pre treated Pos master No status Protein ID Appearance Paired T test Paired Av Ra 1368 1118 Unconfirmed 20 20 A M 0 00085 1 83 1369 1121 Unconfirmed 20 20 A M 0 00074 1 15 1370 1128 Unconfimed 20 20 A M 0 40
250. rol gel Tertiary gt Gal 01 Cy5 Treated gel Cancel In the dialog box that appears browse using the right panel to locate the folder containing the gel images to be processed The image files contained in the selected folder appear in the left panel Click to highlight the image designated to be the primary image in the first gel to be processed conventionally the primary image is the standard image in an experiment employing an internal standard Click the Primary button to select this image as the primary image Repeat the process for the secondary and tertiary image if present using the appropriate selection buttons then click OK The Gel Image Information and Result Files dialog box automatically appears The CyDye DIGE Fluor dyes for the images can be selected in the appropriate pull down menus If the image file names contain the text Cy2 Cy3 or Cy5 these fields are automatically assigned The DIGE labelling chemistry can be selected Min is selected as default The spot co detection process in the Batch Processor generates both a DIA and an XML file for each gel processed The file name of these files can be stipulated in the text boxes in the Results File area of the dialog box Both file names are automatically given identical names and are distinguishable by their file extension It is recommended that the number 1 is appended to the end of the file name of the firs
251. rotein of Interest Ctrl Q Primary image scroll down menu Alt Q Secondary image scroll down menu DeCyder Differential Analysis User Manual 18 1173 16 Edition AA ES Batch Processor keyboard shortcuts File menu Ctrl N New Batch Ctrl O Open Ctrl S Save Ctrl P Print Ctrl B Add DIA Batch Item Ctrl M Remove DIA Batch Item Ctrl D Add Spot Map s Ctrl T Remove Spot Map s Edit Menu Ctrl E Edit Item View Menu Ctrl I DIA Batch List Ctrl V BVA Batch List Process Menu Ctrl R Process DeCyder Differential Analysis User Manual 18 1173 16 Edition AA E 3 269 E 3 270 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA F 1 Related products Item Product Code DeCyder Differential Analysis Software pre 118 1163 05 installed on a PC and a single user licence DeCyder Differential Analysis Software 18 1156 17 including single user licence DeCyder Differential Analysis Software 18 1150 45 additional user licence Ettan Spot Picker 18 1145 28 Ettan Spotter For product information please inquire with your local Amersham Biosciences sales office Ettan Spot Handling Workstation 18 1164 05 Ettan Spot Handling Workstation LWS for 18 1164 06 integration with Ettan LWS Ettan Digester 100 120V 18 1152 59 Ettan Digester 220 240V 18 1142 68 Ettan MALDI ToF Pro 120 V 18 1156 54 Ettan MALDI
252. s Immediately after setting the spot map attributes the Protein Statistics dialog box appears automatically The level of statistical significance in protein differences between the two experimental groups is calculated using the T test In the box entitled Population 1 select Control and select Treated in the box entitled Population 2 Ensure that the Student s T test and Average Ratio check boxes are selected and the Independent tests option button is selected Click on OK Protein Statistics Type of statistical tests Independent tests normal Paired test use sample ID Statistical tests IV Average Ratio opulation 1 Population 2 Dption s Dption s 4 Members 4 Members M OneWay ANOVA between different groups Cancel DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 231 Tutorial IV Fully automated identification of differentially expressed proteins 232 13 5 4 Protein Filter Immediately after setting the protein statistics parameters the Protein Filter dialog box automatically appears The Protein Filter assigns a Protein of Interest and or Pick status dependent on which Filter Action check box is selected to protein spots based on user defined criteria such as Student s T test value average ratio or volume 1 Select the Assign proteins of interest and Assign Pick status check boxes in the Filter Action section of the dialog box For the purpose of this tutorial select t
253. s e g application note 18 1170 83 AA obtainable on the Amersham web site can be denoted by checking the PTM check box 3 6 Customizing display colors The colors used in the various views can be customized by selecting the Colors tab in the Properties dialog box DeCyder DIA Properties Workspace Spot Display Image View 3D View Table View Colors Spot colors Selected Similar Decreased Increased Excluded Pick assigned Picking references and pick locations Histogram colors Histogram data Histogram model H 90 ec Restore to default colors DeCyder Differential Analysis User Manual 18 1173 16 Edition AA DIA Differential In gel Analysis Module Spot colors The colors of spot boundaries displayed in the Image Views and the spot colors in the histogram view can be altered by clicking on the colored circles and selecting a new color Histogram colors The colors of the line graphs in the histogram view can be altered by clicking on the colored circles and selecting a new color Clicking Default restores the original settings 3 7 Saving exporting and printing Save As Workspaces are saved as DIA files dia file extension which contain all the data associated with all the loaded spot maps Select File Save As and browse to locate the folder to save the workspace in Enter a file name then click Save Save Save workspace updates the saved version of the file to include an
254. s Protein Filter or click the protein filter icon The filter allows proteins to either be given a Protein of Interest or Pick Status by selection of the appropriate check box Filter action IV Assign Proteins of Interest I Assign Pick status The general filter settings allows the selection of all detected proteins by selecting the Select all check box This panel within the filter also permits the restriction of the filtering to only a subset population of proteins either those confirmed or those present on a specified number of spot maps General filter settings T Select all IV Restrict to confirmed proteins IV Restrict to proteins present in gt 10 spot maps Proteins can also be selected on the statistical parameters described in section 4 8 The desired statistical analyses check boxes are selected and the desired values can then be entered in the appropriate text boxes DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN Select proteins with A n V Student s T test value Wiis V Average Ratio gt or q W Average Ratio and lt 0 05 xl One way ANOVA value gt 0 lt I Two way ANOVA Time value gt 0 and lt 0 05 Two way ANOVA Dose value gt jo and lt 0 05 Two way ANOVA Interaction value gt fo and lt 0 05 Proteins can also be selected on the basis of their physical properties s
255. s Software BVA Protein Statistics Type of statistical tests Independent tests normal Paired tests use sample ID Statistical tests IV Average Ratio IV Student s T test Population 1 Population 2 Dption s Dption s Control Treated 5 Members 5 Members IV One Way ANOVA between different groups r Calculate Cancel 4 8 2 Defining groups A group in BVA is a collection of spot maps that for the purposes of the experiment cannot be broken down into further sub groups For example 3 replica gels from one sample are considered one group Alternatively 3 gels of 3 different samples treated with exactly the same experimental conditions can also be considered as a group There is no limit on the number of groups that can be assigned in DeCyder Differential Analysis Software BVA The different experimental groups must first be identified in DeCyder Differential Analysis Software BVA in order to undertake statistical analysis of protein expression changes see section 4 5 2 74 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN A 483 Overview of Statistical tests The BVA module has four principle groups comparison or experimental condition comparison methods that can be applied to analyze protein spot data e Average ratio between two groups or two populations of groups e Student s T test Statistical analysis between two groups
256. s have been determined select Process Exclude Filter to display the Exclude Filter dialog box Select the Area and Volume check boxed and enter the found values Click OK Exclude Filter Spot properties IT Filter confirmed spots FT Slope IV Area 50 TT Peak Height gt lt lt lt IV Volume 20000 Current Area of Interest Whole Image Cancel Help An example of a non stringent set of filter parameters that can be used to run a light filter is as follows Area 50 Volume 20000 The four gels used in this experiment were run under identical electrophoretic conditions therefore the filter parameters generated for a representative pair of images performs equally effectively on the images from the other gels in the experiment If comparing gels from different runs it may be wise to perform a single DIA analysis for each run and set filter parameters accordingly Once the gel artifacts have been successfully filtered note the exclude filter parameters then close the DIA module DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 225 Tutorial IV Fully automated identification of differentially expressed proteins 13 5 Processing multiple images Detection and subsequent matching of protein spots from the images derived from the four gels must now be performed Rather than analyzing a large number of individual gels using the DIA module an automated module the
257. s is speeded up When landmarking is completed deselect the Landmark button Note It is usually only necessary to set landmarks on those images that differ significantly from the Master image In addition landmarks may have to be set after matching if there are a high number of wrongly matched spots in which case matching will have to be repeated Often landmarks do not need to be set at all 64 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN The matching process is divided into two steps Level 1 and Level 2 1 The algorithm first matches large spots spread evenly over the whole gel The algorithm matches by comparing positions and sizes of the neighboring spots The matched spots are then used as landmarks equivalent to manual matches The algorithm then starts from these landmarks and matches neighboring spots In this way the matching propagates out from the landmarks by matching a spot and then matching its neighbors 2 Matching is performed by transforming the detected spot center from one gel to the other gel A spot center transformation is considered a match if the transformed co ordinates are close enough to a spot center on the other gel After each matching step is a control that removes obvious mismatches BS To perform matching select Process Match or click the Match icon Select one of the different options to match all the images or just a specific se
258. sed I Excluded spots Picking references and pick locations IT Picked spots Cancel Apply Selecting different protein spots on the images shows that some of the spots that have been detected are in fact either dust particles or artifacts from the gel To remove these artifacts an Exclude Filter must be used on the images 13 4 3 Gel artifact removal A 3 D View clearly shows if a detected spot is a gel artifact rather than a protein spot due to the very steep sides and a pointed top of an artifact compared to the much smoother curve of a protein spot Actual Protein Dust Particle In order to exclude these artifacts from subsequent analysis a set of Exclude Filter parameters must be generated This is the main reason for running the DIA module at this point o 224 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial IV Fully automated identification of differentially expressed proteins In the Table View click on the column header Area until the spot with the smallest area is present at the top of the table Max Slope Area Max Peak Max Volume 2 Scroll down the table from one spot to another until the next spot in the table is an actual protein spot and not a dust particle or gel artifact Make a note of the Area value of the artifact immediately before the real protein spot This value will be used for the Area category in the Exclude filter 3 Repeat this procedure for Volume 4 When both value
259. sented in a gel Gel ID Include sub rankings Maximum Rank 1760023632112339389 1 C Yes No Separate Window All Results No Grouping Excel ust omma Separated Tab Separated O Total number of spots in gel 96 Summary of Gel ID 1760023632112339389 Page 1 Upstream gel information Gel ID 1760023632112339389 Created Second Dimension Electrophoresis 2002 11 11 16 11 1D Strip 1760024629378537448 Fig 8 6 Save the report Detected proteins in gel as XML 2 Click XML in the middle of the dialog to save the file Save it to a location accessible by XML Toolbox DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 149 ED LWS Integration A 8 6 2 Using the Protein Identification tool Zi DeCyder XML Tools Microsoft Internet Explorer File Edit View Favorites Tools Help Q ea Q x ia A gt Search she Favorites necia O A Se we Address C Program Files Amersham Biosciences DeCyder XMLToolbox htm DeCyder 5 0 Ettan Protein Identification Load XML file containing Detected Proteins in Gel LWS standard report Tab Separated Tables G42D DIGE Testfiles EttanProteinidentification files Prote Browse Web Tables Save Options View protein identification data in separate window after saving LWS Pick List save protein identification data without viewing Protein Identification Create Ettan Protein Identification 3 My Computer 1 Either browse for a file
260. ser is then opened at the appropriate address Note The linking is dependent on the place holder string being compatible with the selected database Removing and editing databases Removing and editing are performed in the Database Properties dialog box To remove a database highlight the selected database and click the Remove button To edit a database highlight the selected database and click the Edit button The database can then be amended Click OK to accept the changes DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 97 ED BVA Biological Variation Analysis Module 98 4 13 Viewing protein data Information relating to proteins such as those described in the previous four sections can be viewed through the Protein Table and the Appearance Table The Protein Table displays several proteins simultaneously allowing rapid comparison of data from different proteins The appearance table displays a single protein showing data from individual gels where the protein occurs Data can also be displayed by annotating protein spots in the gel images view which can be a useful tool for presentation of data 4 13 1 Protein Table The table below describes the information contained in the Protein Table Pos Protein Table row number Master No Protein spot number on Master Spot Map Status Confirmation status of protein Protein ID Protein identification for database linking Protein AC Protein
261. sis by excluding the lowest 10th percentile pixel value on the spot boundary 2 Calculate data histogram A histogram is plotted for spot frequency against log spot volume for each spot map 3 Calculate center of volume A Gaussian distribution is fitted to the main peak of the frequency histogram The model curve parameters are optimized to the histogram data using a standard Least Means Square gradient descent algorithm When the optimization is terminated the center of the model curve is denoted as center of volume CoV The CoV therefore represents the central tendency of the spot volume population i e a typical spot volume on specific spot map 4 Calculate normalized spot volumes The normalized spot volume is calculated for each spot on every gel Vnormy V CoV i Where Vnorm is the normalized volume of a spot Vg is the volume of the same spot and CoVg is the center of volume for the spot map DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 5 Calculate normalized spot ratios The ratio of the normalized spot volumes relative to the smallest normalized spot volume for a group of matched spots i e of the same protein is calculated according to Rnorm Vnormy Vnorm min 11 Where Rnorm is the normalized ratio Vnormg is the normalized volume for a specific spot and Vnorm pin is the smallest normalized volume associated with the spot This ratio allows some degree of comparison
262. specified volume by selecting the Volume check box then entering the volume values required The location of the spots on the X and Y axes can also be used for filtering in order that spots near or on the edge of the gel are not selected or to limit selecting too high or low molecular weight proteins The selection criteria for volume and gel region can be based on either the Master gel or the Pick gel if present by selecting the appropriate spot map in the pull down menu DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 113 ER Spot picking 114 After entering the filter criteria select the Filter button to ascertain the number of spots that will be assigned as proteins of interest If the number of resultant spots is unsuitable the stringency of the filter can be adjusted to produce an optimal number of spots for picking Click OK to accept the spot filter parameters Spots assigned as protein of interest are denoted by the presence of the letter I in the Function column of the Protein Table and have the Protein of Interest check box selected The protein of interest status of spots can be removed by selecting Process Unassign all Protein of Interest The preparative gel XML file which has to be first generated in the DIA module section 5 2 can then be imported into the BVA module Select File Import Spot Map s browse to locate the XML file then click Open The preparative gel can then be matched see s
263. t assigned Analysis A and one of the analysis images is assigned as Master M The analysis function designation indicates that each of the images will be included in the statistical analysis The Master function can be assigned to a different image by selecting the image to be assigned as master using the left mouse click then selecting the Master check box at the bottom of the workspace in the area entitled Spot Map Functions The Master function can be assigned to a different image by selecting the image to be assigned as master using the left mouse click then selecting the Master check box at the bottom of the workspace in the area entitled Spot Map Functions IV Analysis A Master M V Template T IV Pick P ja for Spot Map Gellii 21st may_Cy2 Standa All other function assignments are similarly performed 4 5 2 Group assignment A group is a collection of spot maps that for the purposes of the experiment cannot be broken down into further sub groups Groups can include analysis sets such as control groups treated groups time points and temperature points This spot map assignment is necessary to facilitate the inter group statistical analyses in DeCyder Differential Analysis Software The internal standard non experimental group spot maps are assigned DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 59 ED BVA Biological Variation Analysis Module 60 as standard if the word s
264. t 100 Volume 10000 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 167 ED 1 Using DIA module for preliminary investigation of protein changes 168 10 4 5 Ascertaining the 2 S D standard deviation threshold value The 2 S D value calculated for the control control gel is the fold ratio that encompasses 95 of the spots and can be used as a guide to the level of experimental variation This value should be noted and can be set as the threshold mode when analyzing the control treated gel The protein differences being observed on the control treated gel will be above the experimental variation In the Histogram View is a Histogram Selections toolbar with two drop down menus and two information boxes Make a note of the value in the information box entitled 2 S D 10 5 Analysis of control treated gel 10 5 1 Selecting gel images 1 Select File Create workspace there will be an automatic prompt to save the current workspace This is unnecessary in this tutorial therefore click NO to continue 2 Select Double Detection then click OK 3 Browse to locate the folder entitled Tutorial I in the Tutorial data files folder Double click on the image Gel 02 Control Cy3 to select the primary gel image 4 When the load secondary gel image window appears double click on the image Gel 02 Treated Cy5 10 5 2 Spot detection 1 After the images have been loaded select Process Process gel images 2 In
265. t View Process Help D a ost ot Exp Design View Y BVA Exp Gel 04 Cy2 Standard gel Gel 01 Cy2 Standard gel Gel 04 Cy2 Standard gel Gel 01 Cy2 Standard gel Gel 02 Cy2 Standard gel Gel 03 Cy2 Standard gel Group Description Standard Lij Gel 02 Cy2 Standard gel Lij Gel 03 Cy2 Standard gel E Groupi E Group2 E Unassigned Control 1 Gel 04 Cy5 Control gel 1 Gel 01 Cy3 Control gel iij Gel 02 Cy5 Control gel 14 Gel 03 Cy3 Control gel Treated 1 Gel 04 Cy3 Treated gel iij Gel 01 Cy5 Treated gel 1 Gel 02 Cy3 Treated gel 2 Gel 03 Cy5 Treated gel Condition Condition2 a Function for Spot Map Gel 02 Cy5 Control gel Group Sample 1D Spot Map Comment Analysis 4 I Master M I Template T I Pick P Control y Focus Experimental Design NUM 11 5 4 Landmarking Landmarking allows the user to manually define matched protein spots in order to improve the accuracy of the gel to gel matching process when the samples are particularly complex or the gels have not run well 1 In some cases images from different gels may vary sufficiently to require landmarks to be set To set a landmark select the Match Table MT by clicking the MT icon EH 2 Click on the Magnify Image View icon to display the gel images only The image on the left represents the Master image while the image 184 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial Il Employing a
266. t gel 122 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Batch processor 6 Subsequent gels will then be named automatically by incrementing the number at the end of the file name If the number 1 is not appended to the end of the file name then the user is prompted to enter a file name for each gel Click OK when all the data is correctly entered Item 1 Gel Image Information and Result Files Gel Image labels and type Primary Gel 01 Cy2 Standard gel Secondary Gel 01 Cy3 Control gel Tertiary Gel 01 Cy5 Treated gel Chemistry type Result files DIA filename sI XML filename Zo Cancel The Estimated no of spots and BVA Processing dialog box automatically appears The estimated number of spots can then be entered this estimation is used for all subsequent spot map pairs See section 3 3 1 for further details If the gel is a pick gel containing picking references which require autodetection select the check box for Autodetect pick reference markers If BVA inter gel matching is also required select the check box Include in BVA batch list Click OK Item 1 Estimated no of Spots and BYA Processing Estimated no of spots fod 7 Autodetect pickreference markers IV Include in BYA batch list Note If the spot map of the preparative gel image contains the text pick or preparative in the image file name the software will automatically assign the spot map with a Pick status and will
267. t of images Match options Match All Match Primary C Match Pending and Landmarked Cancel Help Match all matches all spot maps in the workspace including those already matched Match Primary matches spot map selected as primary in the Image View Match pending and landmarked matches spot maps that have not been matched and those that have been landmarked after a previous matching process Click Match to commence matching 4 6 3 Match confirmation The matches in the Table View are of two types Auto Level 1 and Auto Level 2 By default the two types of matches are displayed differently in the Image View Auto level 1 and Auto level 2 spots are represented by DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 65 ED BVA Biological Variation Analysis Module Add Match Unmatched Spot Add to Master a darker and lighter spot boundary highlights respectively The boundaries of the unmatched spots are colored red The vector lines in the Image View indicate the positional difference for the same protein spot on different gels The matching can be checked within the Match Table mode to ascertain the accuracy of the matching process This can be done in several ways 1 Confirm a few matches from different areas on the gel by clicking around the image then moving onto the next image recommended option 2 Order the spots in the Match Table by match quality values then confirm the spots w
268. t samples conventional 2 D methods require the separation of each sample on an individual gel This one sample per gel approach exposes the data to a high level of system variation i e the variation that arises from differences in protein uptake into the first dimension strip second dimension gel running etc This high level of system variation can outweigh the often subtle induced biological changes that the experiment is intended to detect for example differences that are caused by a disease state drug treatment or life cycle stage To compound this problem it is also necessary to separate the induced biological changes within an experiment from the inherent biological variation i e the differences between two individual animals cultures plants or flies that are present irrespective of the applied experimental test conditions To achieve this multiple sample replicates must be incorporated within each experimental design This requires the separation and analysis of a large number of samples and can be a slow process if each sample is separated on a different gel Ettan DIGE system has been developed to address these problems The system includes CyDye DIGE Fluor Cy 2 Cy3 and CyS minimal dyes which are mass and charge matched spectrally resolvable fluorescent dyes Additional CyDye DIGE Fluor Cy3 and CyS saturation dyes have been developed specifically to be used where only small amounts of sample are available Please ref
269. t text box DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN AGA Match Quality Metric Match quality values are typically between 1 and 15 expressed as real numbers approximated to two decimal points This value represents morphological but not positional similarities in matched spots The match quality is calculated for only the internal standard spots that are matched across a set of gel images as we know that these proteins should be the same The match quality value is calculated from the surface profile of each internal standard on each gel matched to the master spot maps The surface profiles of all standard spots in a set of matched spots are combined to form an average surface profile Subsequently all the individual internal standard spot surfaces in the match are compared to this normal surface including the spot derived from the master spot map The quality score is the calculated difference between the individual matched spots and the normal spot The spot surfaces that deviate a great deal from the normal surface receive the highest score whereas the spot surfaces that have close resemblance to the normal surface will be given the lowest score Therefore values approaching zero indicate good spot similarity and hence accurate matching As the quality score increases the degree of similarity decreases revealing incorrect matches T
270. tandard pooled or std is present in the image file name By default non standard spot maps are assigned as Unassigned The experimental group assignment is displayed in Experimental Design View when within the Spot Map Table mode Exp Design View F BVA Experiment 3 Standard Y Groupi Y Group2 BE unsssgned 1 JGel 04 Cy3 Treated gel 3 Gel 04 Cy3 Treated gel iij Gel 04 Cy5 Control gel iij Gel 01 Cy3 Control gel iij Gel 01 Cy5 Treated gel LJ Gel 02 Cy3 Treated gel iij Gel 02 Cy5 Control gel 1 Gel 03 Cy3 Control gel ij Gel 03 Cy5 Treated gel I Control Y Treated Grow Unassigned 1 JGel 04 Cy5 Control gel i 2 Gel 01 Cy3 Control gel a l JGel 01 Cy5 Treated gel Description Gel 02 Cy3 Treated gel 1 3Gel 02 Cy5 Control gel Fy JGel 03 Cy3 Control gel Condition 1 JGel 03 Cy5 Treated gel Condition2 Color dk Add Spot Maps are located in the appropriate folder on the left side of the Experimental Design view The Spot Maps can be viewed by selecting the desired group folder Spot Maps can be assigned to the predefined Group1 and Group2 by selecting the Unassigned folder then dragging and dropping Spot Maps from the center panel of the Experimental Design view to the desired folders The names of predefined Group1 and Group2 experimental groups can be renamed by selecting the Edit Name button on the right of the Experimental Design view New c
271. tein spots that have been matched to the protein spots on the master Function Spot Map function e g Master Template Analysis and Pick Group Spot Map group e g Control Treated and Standard Group Description User defined description of group Condition 1 User defined condition assigned numerically example Time point 1 2 3 Condition 2 User defined condition assigned numerically example Dose point 1 2 3 Sample ID User specified identification of sample Comment User defined comment on Spot Map DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 61 ED BVA Biological Variation Analysis Module A 4 5 5 Spot Map Table properties Properties associated with Spot Map Table View are defined in the Spot Map Table Properties window Selecting View Properties or selecting the Properties icon then selecting the Spot Map Table tab displays the Spot Map Table Properties dialog box DeCyder BYA Properties Image View 3D View Graph View Database Colors Spot Map Table Match Table Protein Table Appearance Table Table Column Order and Visibility Select columns to display and the column order by drag and drop Left Column No Status W lmage Gel No Type Label Y No of Spots W Matched v Function v Group Group Description v Dose Y Time Sample ID Y Comment Match Quality Right Column Restore to default order and se
272. tein spots with a volume ratio greater the 2 SD value from the control control gel and a max volume between 10 and 10 for example Click on Filter to calculate the number of spots that meet the criteria These values can be adjusted and the Filter button clicked again to see how many spots now meet the criteria The purpose of this filtering is to narrow down the users search for interesting proteins that are significantly changing Click OK DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial Using DIA module for preliminary investigation of protein changes 10 Protein Filter Filter action IV Assign Protein of Interest I Assign Pick status General filter settings I Restrict to Confirmed spots Select spots with IV Volume Ratio or IV Volume Ratio Area IV Max Volume gt 1 008 005 and lt 1 00e 008 T Max Peak Height gt 20000 and lt 100000 TX co ordinate gt n and lt foo M Y co ordinate gt ot and lt foo Filter Cancel Help 4 The subset of spots that met the criteria will now be assigned with a protein of interest in the protein table denoted by the letter I in the Function column 5 Select View Properties or click on the Properties icon Go to the Table View tab and select the Protein of Interest check box and uncheck all of the other options The table view will now display only those spots assigned as protein of interest in the protei
273. the process gel images dialog box which now appears enter the value 2500 for the estimated number of spots see help file for an explanation of this value 3 Click OK to begin spot detection on the images 10 5 3 Spot filtering 1 Assign an Area of interest and Filter as described for the control control gel using the same filter parameters DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial Using DIA module for preliminary investigation of protein changes 10 E 1054 Setting a threshold 1 Using the Threshold Mode drop down menu in the Histogram Selections toolbar of the Histogram View thresholds can be set to highlight protein spots that differ in their abundance For example to select spots whose volume ratios differ between samples by 2 fold or more select 2 0 fold from the drop down menu Histogram selections Scatter parameter Max Volur Threshold mode 2 0fold Threshold 2 25 D 1 45805 A variety of values in the Threshold Mode box can be selected such as 2 S D which is based on the model histogram blue line in the Histogram View Alternatively the threshold mode can be set manually by selecting Manual and then entering a figure in the Threshold information box Enter the 2 S D value ascertained in the control control gel into the information box As previously discussed this value represents the inherent experimental variation It can therefore be hypothesized that spots with
274. the top of the table to see whether the spot with the largest volume has been sorted to the top of the table if the spot with the lowest volume is at the top of the table click once more on the max volume header The spots with the largest Max volume and hence at the top of the sorted table are almost exclusively protein spots which can be quickly confirmed Scrolling down the table to spots with lower Max volumes non proteinaceous spots start to appear which can be manually excluded Spot confirmation pertains to a Spot Number therefore when using triple detection a confirmed spot number will possess the confirm status irrespective of which spot maps are displayed in the image view SY DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 47 DIA Differential In gel Analysis Module 48 3 5 3 Protein of Interest Protein spots can be assigned as a Protein of Interest thereby highlighting these spots for further investigation Protein of Interest status can be assigned manually by selecting the Protein of Interest check box when highlighting the desired protein spot I Pick FT PTM 5 Confirm IV Protein of Interest J Exclude Alternatively the Protein filter can be used to automatically assign Proteins of Interest using spot property as a selection criteria 3 5 4 PTM assignment Protein spots possessing a post translational modification PTM identified by methods outlined in Ettan DIGE system application note
275. ting File Save As files are saved as text files The batch list text files can then be re opened later by selecting File Open The folder that the DIA and XML files generated when processing the DIA batch list can be selected by clicking the Browse buttons adjacent to the respective directory text box Similarly if the BVA batch list is also performed the names and destinations for saving the subsequent BVA file and pick list if requested can be stipulated by selecting the Set buttons adjacent to the respective file name text box File Edit View Process Help DIA Dir c Fiman Manual workspaces Spiking experi Browse NBVA Filename e WFiimaniManual workspaces Spiking exp XML Dir e Firman Manual workspaces Spiking experi N Browse A Pick Filename C Fiiman Manual workspaces Spiking exp DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 127 6 Batch processor 6 5 Running the batch processor To run the Batch Processor select Process Run batch If the default BVA filename has not been changed the user will be given the option to change this The Run batch dialog box which now appears gives the user the option to begin processing immediately or introduce a delay period The batch processor must be left open during the delay period To begin the countdown to processing or to start processing click OK FE DeCyder Batch Processor batch bd DIA Dir Je Firman Manual wo
276. tion1 Two way ANOVA for condition 1 p value for condition 1 ANOVA2 condition2 Two way ANOVA for condition 2 p value for condition 2 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA XML Toolbox ANOVAZ2 interaction Two way ANOVA interaction p value for the interaction between condition 1 and condition 2 Paired T test Paired Student s T test p value Paired average ratio Paired average ratio between the groups selected for protein analysis RM ANOVA1 Repeated Measures ANOVA Paired One Way ANOVA p value RM ANOVA2 condition1 Paired Two Way ANOVA p value for condition 1 RM ANOVA2 condition2 Paired Two Way ANOVA p value for condition 2 RM ANOVA2 interaction Paired Two Way ANOVA p value for the interaction between conditions 1 and condition 2 7 4 2 DIA parameters Experimental data Primary Image Refers to the left hand gel image present in the image view Secondary Image Refers to the right hand gel image present in the image view DIA experiment Gel name designation No of included spots Number of spots designated as included for subsequent analysis Dye chemistry Indicates whether minimal or saturation labelling was used Estimated no of spots User defined approximation of the number of spots present in spot map image entered prior to spot detection No of similar spots Number of spots within the threshold mode set No of increased spots Number of spots showing an
277. ttings I Link image views when scrolling IV Auto center selected spots Picking Reference Data Picking references and pick locations Primary Image View Default radius of picking references 5 100 Default diameter of picker head 5 50 Secondary Image View Default radius of picking references 5 100 Default diameter of picker head 5 50 Cancel Apply Include in Image View Select check box to display only the spots displayed in the Table View Signature Select to display the spot boundaries Annotation Select to display user determined annotation as text boxes on the gel Image Views The annotation displayed is selected from the list only one category can be selected Selecting the Link Annotation check box results in spots derived from different isoforms of the same protein defined by the user by having the same Protein ID AC name or comment being grouped and labelled with a single label The Filter Annotation box can be selected to only display annotations for pick status spots or spots selected as proteins of interest DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 55 ED BVA Biological Variation Analysis Module 56 Match vectors when Match Table is displayed Select to display the match vectors see section 4 6 2 Link Image Views when scrolling Select to link scrolling in both images Auto center selected spots Select to position selected spots in the center
278. tube generated within Ettan DIGE system into Ettan LWS The workflow for the samples should be set up the normal way in Ettan LWS The only differences are within sample attributes template and sample definition A user defined sample attribute template must be set up to match the experimental design within Ettan DIGE system By creating a DIGE sample template in Sample Attribute Templates it will be possible to later search for the information and to produce Ad hoc reports For information on Ad hoc reports please consult Ettan 2D MS and LWS software Appendices 1 Inthe Management Workbench Sample Template Editor define a sample type for a sample generated within Ettan DIGE system by adding user defined attributes An example is shown in Fig 8 3 sample Attribute Templates xi Application Ettan 2D MS Sample Types for Ettan 2D MS Templates for Protein All Sample Types Core Template for Protein El al Bluebird Crane Add Copy Edit y Attributes for DIGE Default Is Required Fig 8 3 Example of attributes for a sample type generated within Ettan DIGE sys tem The samples need to be defined in ScierraTM web 2 In Scierra web select project name and Define Samples 3 Scan or type sample ID choose Sample Type Protein Sample 142 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA LWS Integration 8 Template the user defined template
279. twork file server e Background noise Background is defined as undesired signal often resulting from autofluorescence or light scatter from a matrix or sample support It can be minimized by the selection of appropriate matrix or sample support e g low fluorescence glass Noise is defined as the statistical uncertainty inherent in a measurement such as the standard deviation associated with measured background counts e g with 2 D gels noise can be attributed to contaminants with fluorescent properties similar to the specific fluor being used By ensuring that only high quality reagents are used and recommended procedures are followed noise can be minimized The sensitivity of the imaging system can be adjusted by changing exposure times for CCD based systems or voltage settings for PMT based systems The specific signal can be optimized to give the highest signal to noise ratio thus ensuring the maximum amount of information is obtained from the image DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 249 250 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Appendix C Spot processing algorithms C 1 DIA normalization The DIA normalization algorithm represents a series of steps that allows spot quantitation to be expressed as a function of the primary image where an unchanged protein is represented as zero Therefore when using an experimental design employing an internal standard all the analysis
280. ubjected to 2 D gel electrophoresis The samples from the other 4 volunteers were similarly subjected to 2 D gel electrophoresis on separate gels as described in the table overleaf standards on individual gel not shown DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 257 Spot Map Table 258 Matched Master Matched Matched Matched Matched Matched Matched Matched Matched Matched Matched Matched el 01 al Gel 01 Cy5 gel Gel 02 Cy2 Standard gel Gel 02 Cy3 gel Gel 02 Cy5 gel Gel 03 Cy2 Standard gel Gel 03 Cy3 gel Gel 03 Cy5 gel Gel 04 Cy2 Standard gel Gel 04 Cy3 gel Gel 04 Cy5 gel Gel 05 Cy2 Standard gel Gel 05 Cy3 gel Gel 05 Cy5 gel Assigning groups and individuals in DeCyder Differential Analysis Software Spot data can be assigned into two experimental groups pre and post drug treatment represented by 1 and 2 respectively The same 5 volunteers are present in both groups therefore the data can be paired i e assignment of individual volunteers within groups The CyDye DIGE Fluor Cy2 minimal dye labelled internal standard samples are omitted from the table below Number Gel CyDye Individual Group 1 1 Cy3 1 1 2 1 Cy5 1 2 3 2 Cy3 2 2 4 2 Cy5 2 1 5 3 Cy3 3 1 6 3 Cy5 3 2 7 4 Cy3 4 2 8 4 Cy5 4 1 9 5 Cy3 5 1 10 5 Cy5 5 2 The above parameters can be designated either in the DeCyder Differential Analysis Software Batch Process
281. uch as volume X co ordinate and Y co ordinate These physical properties pertain to the spots on the Master spot map or the Pick spot map if present depending on which is selected from the pull down menu Properties for proteins in spot map I Volume gt and lt TX co ordinate gt and lt TY co ordinate gt ft and lt Note The use of the protein filter in selecting spots for picking is further discussed in section 5 4 10 Molecular weight Mw and isoelectric point pl DeCyder Differential Analysis Software BVA provides the user with the functionality to calculate and display the molecular weight Mw and isoelectric point pI of the proteins on all the images being analyzed The Mw and pl of known proteins can be entered into the Protein Table these values known as the base list are then used to calculate the Mw and pl for all the other proteins on the images 4 10 1 Entering Mw and pl of known proteins To enter the Mw and pI of the known proteins in your experiment select the protein in the Protein Table PT and enter the Mw and pl into the Mw and pl text boxes in the data control panel The Mw and pl entered for a protein can be edited by clicking on the protein and changing the values in the Mw and pl boxes DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 91 ED BVA Biological Variation Analysis Module calculating Mw and pl ny To calculate the Molecular weight Mw and isoele
282. uirements coooooocccccncconcnnnnnnoncnnnnnnononnnnnnncnnonnnnnons 23 CONAN cari ai a a bras topes waste tect tac 87 Consumables ura ai thet hug vd on te Loa DOT mee 272 Contrast and Brightness ccccnnnnocecocooooonnonononnnnnnonononanananananananos 34 Create A 29 53 Customizing display Colors cccccncnccccnccncononcnononinonanononos 48 103 CyDye DIGE Fluor minimal dyes ococooccccccconoccnccccononanononononanonoss 12 D Data Control Panel artists a Al ake ens 28 Database linking oooocccccccccooococncnnnnnnnnococononononnononanonn nono nonnnnnnnnoss 94 yA DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 279 280 Database query in DeCyder BVA cccccnncccccccconoonnnncnnononnnncnonnn 151 DeGyder XML Citi e di ide 129 DeCyder XM TOODO irae atie n iada ea 140 EWS Pick Listtool iii ia 146 Protein Identification tool 140 148 Detected proteins in gel ee eeeseeeeeesenneeeceesesneneceesesseneeeers 149 Aes lt o dd ER 27 155 159 DIA Graphical User Interface occccccccccccnnncnninononanananannnnnnnna 28 DIA Module total dui Steet 27 DIA PROCESSES su tati didas ZY Digital Mages na lt eo ista 243 Display Mw and pl values oooooccccccnnnnccccnnnonononononininnnnanannnnnnnnos 93 DUSEPATUCIOS cu ada A A ele eee 44 Dynamic fange serii ht tado 248 E Enter Mwand Pi a aia 91 Enter samples into Ettan LWS ooooccccccncccccccoconoonnononononanonononos 140 aS a Gel A suriati at eal
283. undary colors of the different categories of spots displayed in the Match Table Image View can be changed by clicking on the colored circles then selecting a new color Spot Colors Spot boundary colors of the different categories of spots DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 103 ED BVA Biological Variation Analysis Module 104 displayed in the Protein Table and Appearance Table Image Views can be changed by clicking on the colored circles then selecting a new color Annotation Colors The colors of the gel image annotation text boxes and linking lines can be changed by clicking on the colored circles then selecting a new color Spot Map Colors The colors that designate different categories of spot map in the Image Views can be changed by clicking in the colored circles then selecting a new color The original settings can be restored by clicking Default 4 13 5 Saving exporting and printing Save As Workspaces are saved as BVA files which contain all the data associated with all the loaded spot maps The BVA files do not contain the image files but do contain information on their file path Select File Save As and browse to locate the folder to save the workspace Enter a file name then click Save Save Save workspace updates the saved version of the file to include any changes that have been made to the workspace DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biol
284. ure that only these protein are displayed In Appearance Table mode select the first Protein of Interest Review the Protein of Interest across all analytical gels to check the matching accuracy Use the Add Match or Break Match No functions to add amend matches Isthe matching accurate In Match Table mode usethe Add Match or Break Match functions to add amend matches Yes Confirm Protein Select the next Protein of Interest ls there another Protein of Interest No BVA file for whole experiment containing confirmed prtoeins of interest DeCyder Differential Analysis User Manual 18 1173 16 Edition AA A 3 Flow Diagram 3 BVA file for whole experiment containing confirmed proteins of interest Wasthe preparative gel processed in the Batch Processor No y Open DIA module and select File create workspace J Select Single Detection and load the preparative gel image Detect spots by processing gel image using estimated number of spots Ensure the Autodetect Picking References check box is selected Click OK i XML file for the preparative gel Select File Export Spot Maps BVA file for whole experiment Open BVA module amp select File Open W orkspace to open BVA generated in Batch Prooc
285. ure that the image on the right is the SYPRO image using the Select Match Gel scroll down menu To begin setting landmarks click the button entitled Landmark mode at the bottom of the screen this button will now become yellow All the spots should appear red to indicate that they are unmatched If the spots on the images are not clearly visible it may be necessary to alter the contrast brightness settings of the images Click on the DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial III Processing the Preparative Gel and Generating a Pick List 12 contrast brightness icon ensuring the Apply to all gel images check box is not selected Alter the position of the bars to alter the contrast and brightness of the images until only the most intense spots are visible 5 To set a landmark click on a clearly defined spot on the master image the spot boundary should become magenta Now select the spot on the match image which corresponds to the master image spot so that this spot becomes magenta A few seconds later a vector line should appear showing that the spots have been matched and the landmark has been set Fi DeCyder BYA E coli control treated bva File Edit view Process Help j GE stir errar Y 3 Match Image Master Spot Landmark mode Unmatched Spot Master Spot Match Comment ay E JO eaf at anea tono Focus Master Image NUM 6 Set approximately 10 20 landmarks on t
286. used to investigate differential protein expression in experimental groups These identified proteins can then be picked from a preparative gel for digestion and subsequent mass spectrometry analysis Gels 1 to 4 were generated to ascertain those proteins that were differentially expressed in control and treated bacterial samples These gels have been pre analyzed in DeCyder Differential Analysis Software and saved as a BVA file 198 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial III Processing the Preparative Gel and Generating a Pick List 12 E A SYPRO Ruby stained preparative gel gel 5 loaded with both control and treated samples was prepared for spot picking The spots on this gel are first detected within the DIA module then matched to the analytical gels gels 1 to 4 in DeCyder Differential Analysis Software BVA Gel number Cy2 Cy3 Cy5 SYPRO Ruby Gel 1 Standard Control 1 Treated 1 Gel 2 Standard Treated 2 Control 2 Gel 3 Standard Control 3 Treated 3 Gel 4 Standard Treated 4 Control 4 Gel 5 Control Treated 12 3 1 Identifying protein spots on SYPRO Ruby stained gel 1 Double click the DeCyder Differential Analysis Software DIA icon a on your desktop to open the DIA module 2 Select File Create Workspace File Ei View Process Help Create Workspace Ctrl N Open Workspace Ctrl 0 3 Select Single Detection then click OK Type of Detecti
287. ustom named groups can be generated To create new groups select Add then type in the desired name of the group in the Group box A new group folder is subsequently displayed The assigned groups are also displayed in the Spot Map Table in the Group column The description text box adjacent to the Group assignment can be used to enter user defined information regarding the description of the experimental group This description is linked to the experimental group DeCyder Differential Analysis User Manual 18 1173 16 Edition AA IV Analysis A BVA Biological Variation Analysis Module EN 4 5 3 Comment assignment The comment text box on the right of the data control panel can be used to enter a user defined comment that is specific to the Spot Map in the primary view a Function for Spot Map SYPRO gel FT Master M Template T Pick P Group Sample ID Spot Map Comment Unassigned 4 5 4 Spot Map Table Mode The Spot Map Table Mode displays data associated with the spot maps This data is derived from the XML file generated in the DIA module No Spot map number in table Status Indicates whether the image is Matched Pending or Processing Image Gel image file name Type Type of labelling chemistry used Label Dye used to label the protein e g CyDye DIGE Fluor Cy2 Cy3 and Cy5 minimal dyes No Of Spots Number of protein spots identified in the DIA Matched Number of pro
288. ut table For XML files exported from BVA four selections are available Picked proteins Proteins of interest All matched proteins and All proteins For XML files exported from DIA only the Picked proteins and All proteins selections are available Type of proteins in XML file to map onto the row of the tables Picked proteins O proteins of interest Oall matched proteins Oall proteins DeCyder Differential Analysis User Manual 18 1173 16 Edition AA XML Toolbox A Category of data in table columns The data is categorized into four groups 1 Experiment data The table contains general experimental set up parameters valid for the current DIA or BVA experiment 2 Image data The data in this table contains the different parameters that are unique to each spot in the individual gel images contained in the selected DIA or BVA experiment 3 Gel data The data displayed in this table is unique for each detected spot pair within each set of gel images that originate from the same gel 4 Protein data All protein data that is unique to a set of matched spots across the different gels in the current BVA experiment is displayed in this table Category of data to map onto table columns Experiment data vi Experiment data Image data Gel data Protein data The interfaces diverge when selecting categories of data e Web tables Each of the four lists of data items represents a type of result table Thus if at least
289. ve the old version and automatically install the new version Click Next Note If DeCyder Differential Analysis Software version 4 or earlier is already installed the installation process will continue as a de novo installation see section 2 2 2 automatically removing the previous version of the software DeCyder Differential Analysis Software Ti The DeCyder Differential Analysis Software is allready installed on this computer Please select whether to reinstall the DeCyder software again or to remove it from your system by clicking the appropriate option button below Reinstall 1P Reinstall all DeCyder components installed by the previous setup Remove El Remove all installed DeCyder components coca DeCyder Differential Analysis Software can be un installed automatically by selecting Remove all installed DeCyder components 24 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Computer requirements and installation 2 E 222 De Novo Installation If DeCyder Differential Analysis Software has not been previously installed on the system a different dialog box will appear when the Setup icon is selected Click Next to install DeCyder Differential Analysis Software DeCyder Differential Analysis Software DeCyder Differential Analysis Software Installation Welcome to the installation of DeCyder Differential Analysis Software In the subsequent dialog box a default file path for the inst
290. ware DIA graphical user interface is divided into four equally sized inter linked views e Image View primary and secondary gel images e 3 D View a three dimensional representation of the gel localized on the spot e Histogram View graphical representation of data associated with the spots displayed in the image view e Table View tabulated data associated with selected spots displayed in the image view All four views are linked therefore selecting a spot in for example the Image View will display the spot in the Histogram View in magenta the 3 D View and the Table View The role of the four views will be discussed in detail later in this section Below the four views the Data Control Panel is found This panel contains tools and user definable functions associated with the specific data displayed in each mode fl DeCyder DIA Control Treated d a File Edt View Process Help Os Ont ke He 992 Beso a Sw Secondary Gel 02 Treated v 2 Histogram selections laa r B Scatter parameter Max Volur_ Primary Gel 02 Control Gy3 v E Threshold mode 2 model S x Threshold 24551 25 D 232553 Spot istics Decre d 116 4 8 Similar 2235 91 6 Increased z 90 3 7 A ws Histogram View Detected 2441 Included 0 7 Excluded Log Volume Ratio Picked Spot No Abundance Ex Volume Ratio Picked 1 92 Un z ilar
291. workspace Ctrl P Show print dialog Edit menu Ctrl T Add Spot to Table Ctrl C Copy View menu F7 Zoom In FS Zoom Out F9 Fit to window Ctrl B Contrast Brightness Ctrl A View Area in 3 D Ctrl R Rotate 3 D Help menu Shift F1 Help What s This DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 267 E 2 BVA module keyboard shortcuts Workspace Alt F Open File menu Alt E Open Edit menu Alt V Open View menu Alt R Open Process menu Alt H Open Help menu File menu Ctrl N Create new workspace Ctrl O Open workspace Ctrl S Save workspace Ctrl P Print View menu F7 Zoom In F8 Zoom Out F9 Fit to window Ctrl B Contrast Brightness Ctrl A View Area in 3 D Ctrl R Rotate 3 D Alt Enter Properties Modes Alt S Spot Map Table Alt M Match Table Alt P Protein Table Alt A Appearance Table Help menu Shift F1 Help What s This Protein Related functions Alt 1 Select next row in current table Alt 1 Select previous row in current table Alt Page Down Scroll down current table Alt Page Up Scroll up current table Alt gt Select next Spot Map protein Alt Select previous Spot Map protein Alt X Set focus to Controls Alt C Confirm Unconfirm protein or match Alt D Add Break match Alt K Pick protein Alt P
292. x y co ordinates of spot positions is then exported based on the preparative gel The pick list along with the physical preparative gel is then taken to Ettan Spot Picker for spot excision and later analysis by mass spectrometry For a detailed description of the workflow associated with pick list generation see Chapter 3 Generating a pick list is covered in more detail in Tutorial III DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 195 ED II Employing an internal standard to Analyze Protein Changes 196 DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Tutorial III Processing the Preparative Gel and Generating a Pick List 12 12 Tutorial Ill Processing the Preparative Gel and Generating a Pick List 12 1 Objective As can be seen in tutorials I and II proteins of interest which warrant identification and further investigation can be obtained in both the DIA and BVA modules The Protein Filter tool is applied to the analytical gels to rapidly ascertain the proteins that are affected by the experimental conditions When using either CyDye DIGE Fluor minimal dyes or CyDye DIGE Fluor saturation dyes the highlighted proteins of interest have to be matched to a preparative gel from where the protein spots can be directly excised using Ettan Spot Picker or Ettan Spot Handling Workstation see Ettan DIGE system user manual The workflow for generating a pick list from a preparative gel in both CyDye DI
293. y changes that have been made to the workspace Printing Select File Print to open the Print dialog box Print Image View Histogram View Y Primary Image MV Secondary Image IV Histogram View V Both side by side 3D View Table View IV 3D View M Table View Number of copies General Information j General Information JA 2 Printer Name MAPBAMEOSSHP LaserJet 4050 Series PC_w Properties Status Ready Type HP LaserJet 4050 Series PCL 6 Where LPT1 Preview Cancel DeCyder Differential Analysis User Manual 18 1173 16 Edition AA 49 DIA Differential In gel Analysis Module 50 Multiple check boxes can be selected for printing the required workspace views DeCyder Differential Analysis Software software supports the following printers e HP LaserJet 4000N HP color LaserJet 5M e HP 2500C e HP DeskJet 895 Cxi e HP DeskJet 950C e HP Laser Jet 4100 DIN Note Other manufacturers and models of printers have been demonstrated to be compatible with DeCyder Differential Analysis Software Using the clipboard Any aspect of the different views within the workspace can be copied to the clipboard for pasting into another application Click on the part of the workspace chosen for pasting then select Edit Copy the part of the workspace selected is described in this pull down menu The clipboard can then be pasted into other applications Exporting data Each of the export functions in DIA is l
294. y created workspace should now be saved see section 4 13 5 4 3 2 Opening workspaces To open previously created and saved workspaces select File Open and browse to locate the required BVA file When the file is located select the file and click Open 4 4 Viewing spot data The general toolbar functions associated with the Image 3 D and Table View are identical to those in the DIA and are described in sections 4 4 1 4 4 2 and 4 4 3 respectively 4 4 1 Image View Simultaneously displays two gel images The gel image displayed can be selected using the pull down menu in the title bar of each gel image window DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN LES Properties Properties associated with Image View are defined in the Image View Properties window Selecting View Properties or selecting the Properties icon and then selecting the Image View tab displays the Image View Properties dialog box DeCyder BYA Properties Spot Map Table MatchTable Protein Table Appearance Table Image View 3D View GraphView Database Colours Include in Image View I Spots present in table only Spot Features IV Signature IV Annotation Master Number Protein ID Protein Name Protein Comment pl and Mw Protein of Interest Link Annotation I Filter Annotation ERA IV Match vectors when Match Table is displayed Scrolling and Auto Center Se
295. y automated co detection quantification and matching of an in gel image pair DIA Batch List Spreadsheet in the batch processor depicting which image pairs are to be detected and quantified Exclude Filter Filter used to remove non proteinaceous artifacts such as dust Groups The collection of spot maps relating to image pairs whose sample images have undergone exactly the same experimental conditions e g 3 spot maps of samples from disease patients treated with drug A at a specific dose is a single group Histogram Selections User adjustable parameters that alter the appearance of the histogram in the DIA module Independent Data Data sets which have no effect on one another Label Indicates the fluor used to label the protein Landmarking Process of manually matching spots to the master image before automated matching to aid the matching algorithm Master Image Spot map to which all others are matched to Match confirmation Manual verification by the user of matched spots Matching Automatic linking of spots on selected images to the corresponding spot on the master image Match table Area within a BVA workspace where spot match data is displayed DeCyder Differential Analysis User Manual 18 1173 16 Edition AA Maximum Peak height Pixel value at the X Y position of the spot This is the actual detected peak height value compensated for background level Maximum Volume The highest volume from two co detecte
296. y of obtaining the observed data if the groups have the same protein abundance For example if the ANOVA p value between two groups is 0 01 then the probability of obtaining the observed difference in protein abundance by stochastic variation alone is 1 in a 100 Protein abundance differences are generally assumed to be statistically significant when p lt 0 05 However a critical value of 0 05 would mean that 5 of the data points would be expected by stochastic events alone e g 50 spots out of 1000 tested will be false positives It is therefore advisable to review the critical value applied to the data Due to the small amount of experimental variation within Ettan DIGE system a critical value of 0 01 is often applied Assumptions and limitations The main assumptions of the ANOVA tests are the following e The populations from which the samples were obtained are normally or approximately normally distributed e The variances of the populations must be equal There is a certain amount of leeway in both these assumptions particularly the first small departures from a normal distribution are DeCyder Differential Analysis User Manual 18 1173 16 Edition AA BVA Biological Variation Analysis Module EN unlikely to be of any consequence However if the variance among the groups differs greatly the ANOVA p value may have poor validity 4 8 7 One Way ANOVA Overview The simplest form of ANOVA is known as one way ANOVA and in DeC
297. y visible when more than one image is being analyzed i e analyses employing either double or triple detection algorithms Clicking on the Histogram View icon or selecting View Histogram View Y ds the Hi iew to fit the work expands the Histogram View to fit the workspace Fl decyder Dia D d ur BESS ER al vens re 197 1 Threshold 25 250 3 81045 4 887 1 2E7 DES Spot statistics 157 Decreased 56 2 5 Simlar 1884 183 6 Increased 314 13 9 HES Workspace information 4 267 Detected 2254 Included 2254 Excluded o Picked o 1 487 ANE 187 OA x El Protein ID Comment Confirm El T Protein of Interest 7 Exclude Display all views Histogram The histogram displays data associated with all detected spots in the primary and secondary images Spot data is plotted against log volume ratio on the X axis using two Y axes e Left Y axis displays the spot frequency The red curve represents the frequency distribution of the log volume ratios The curve in blue represents a normalized model frequency fitted to the spot ratios so that the modal peak is zero see Appendix C e Right Y axis represent the scatter parameter selected in the histogram selection box right of the histogram A plotted single data point on the histogram represents an individual protein spot The histogram is automatically recalculated when the primary an
298. yder Differential Analysis Software is used to test for differences in standardized abundance For example a clinical trial might consist of three groups of patients according to whether they were given drug A drug B or placebo ANOVA could be used to test the null hypothesis that there is no difference in standardized protein abundance among the groups The test will not indicate which groups are different from which other groups just that there is an overall difference In the above example a significant difference does not necessarily mean that drug A is different from drug B or that either drug is different from placebo All a One Way ANOVA test indicates is that in someway the patients are affected by one of the drugs Performing analysis 1 In the Protein Statistics dialog box select the option button to indicate whether the test is independent or paired A paired ANOVA test is called a repeated measures ANOVA RMANOVA Pairing of spot maps have to be pre assigned in the spot map Table View see section 4 8 4 for paired testing 2 Select the One Way ANOVA check box all assigned groups are included in the One Way ANOVA test Click Calculate 3 Select the Properties icon on the tool bar and ensure that 1 ANOVA check box is selected in the Spot Map Table tab If the One Way ANOVA analysis is paired select the Paired tests option button A repeated measures paired One way ANOVA will then be automatically calculated Click Cal
299. yder Differential Analysis User Manual 18 1173 16 Edition AA 133 XML Toolbox 134 Match status Indicates whether match has been confirmed Spot number DIA spot number as given by the spot detection algorithm Protein data Picked status Indicates whether the protein has been assigned for picking Protein ID Unique protein identifier that can be used to search databases providing it is in the correct format for the specific database Protein AC The protein accession number as designated by the selected database Protein Name Protein name given by selected database Protein comment Note typed in the protein comment box Protein function Proteins assigned Protein of Interest are marked with an I in this column pl Isoelectric point of the protein pl Status Indicates whether displayed isoelectric point was entered for reference purpose of the algorithm calculated from the selected algorithm or manually edited designated Template Calculated and Manual respectively Mw Molecular weight of protein Mw Status Indicates whether displayed molecular weight was entered for reference purpose of the algorithm calculated from the selected algorithm or manually edited designated Template Calculated and Manual respectively T test value Student s T test p value Average ratio Average ratio between the groups selected for protein analysis ANOVA1 One way ANOVA ANalysis Of VAriation p value ANOVA2 condi
300. ysis Software analysis a critical value of 0 01 is often applied Assumptions and limitations The main assumptions of the Student s T test are the following e Log standardized abundance values within a group are normally or approximately normally distributed When the assumption of normality is violated the T test tends not to perform well when the sample size is small or the significance level is small e g p lt 0 01 However two tailed tests are surprisingly robust even for skewed data e The parametric variances of the two groups are equal However the test tends to be robust when the sample sizes are approximately equal If the sample sizes are not equal then the worst case occurs when the smaller sample comes from the population with the larger variance Performing analysis The user can perform T test calculations and average ratio calculations between any two groups or populations of groups 1 In the Protein Statistics dialog box select the option button to indicate whether the T test is independent or paired Pairing of spot maps has to be pre assigned in the Spot Map Table mode see section 4 8 4 for paired testing 2 Select the two groups in the in the population 1 and population 2 lists population of groups can be selected by ctrl left mouse clicking the selected groups Select the Average ratio and the Student s T test check boxes Click Calculate R DeCyder Differential Analysis User Manual 18 1173 16 Editi

User Manual DeCyder Differential Analysis Software, Version 5.0

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