Lipid Data Analyzer 1.6
Contents
1. Zoom ali Save 20000000 oo Determine Area so00000 Betermine Area ol z Determine Area reed Determine Ares ae Enter probe borders Delete Area If the mouse is hovered over the painting canvas a crosshairs cursor is rendered whereupon the current time and intensity 1s displayed in the two text fields on the right side of the canvas e g t 23 33 and Int 7 456e6 The third text field indicates the m z value of the chromatogram With Gain the displayed intensity range can be zoomed and with lt lt and gt gt it is possible to shift the currently zoomed time range If a peak is quantified and stored it 1s displayed in red if it 1s just quantified and not stored it s displayed in green If the right mouse button is clicked under a curve the displayed popup menu appears with the following options e Determine Area detects a peak area with the standard ASAPRatio algorithm e Determine Area Col detects a peak area with the MASPECTRAS algorithm that integrates over local maxima e Determine Area Greedy sets the peak borders at sudden changes in the steepness of the curve e Determine Area 3D 3D algorithm for more accurate peak border confinement e Delete Area deletes a quantified area e Enter probe borders manually define the borders of the peak Sl borders X Enter the borders of the peak start time 21 442 min stop time 23 442 min The peak can be defined2. Ida genome tugraz at 24
3. see chapter 5 1 number 5 are required weight end volume returns the weight gram in the end volume before the MS measurement For this value standardization on an internal standard and absolute quantities see chapter 5 1 number 5 are required amount sample volume Returns the amount mol in the sample volume before the sample preparation steps dilution For this value standardization on an internal and or external standard and absolute quantities see chapter 5 1 number 5 are required conc sample volume Returns the concentration mol L in the sample volume before the sample preparation steps dilution For this value standardization on an internal and or external standard and absolute quantities see chapter 5 1 number 5 are required weight sample volume Returns the amount gram in the sample volume before the sample preparation steps dilution For this value standardization on an internal and or external standard and absolute quantities see chapter 5 1 number 5 are required relation to measured neutral lipid This is for the standardization on the total measured lipid mass g of the current lipid class measured by MS For this value standardization 13 on an internal and or external standard and absolute quantities see chapter 5 1 number 5 are required relative to sample weight This is for the standardization on the total weight of the sample mass g of the current lipid class measured by MS For this value
4. the m for milli is defined by this setting This setting is applicable for conc end volume conc sample volume relation to measured neutral lipid relative to sample weight relation to protein content and relation to neutral lipid content o use AU use arbitrary units instead of SI units e Select molecules To fade out specific molecules of the heat map e Combined chart Shows selected molecules in one chart over all samples groups see chapter 5 3 e Export options Defines the information that is exported to Excel or text format This dialog box consists of a radio button with the options analytes in column and experiments in column and additionally a checkbox for the export of the retention time Additionally for groups the standard deviation or the standard error of the values and the standard deviation of the retention time can be exported 5 3 Bar charts The bar charts are accessible via the heat map or the Combined chart button see chapter 5 2 14 FA Lipid Data Analyzer 1 6 0 FT settings Bl x Quantitation Batch Quantitation l Statistical Analysis Display Results Settings License I Help Selection li TAG Heatmap Bar chart Group Heatmap Group bar chart quant tyne absolute quantity NaN ed double sided logarithmic linear standard deviation 1 0 standard error mean 2 E export deviation val
5. 5 58 7 58 11 58 12 LJ 58 13 LJ 60 1 L_ 60 2 _ 60 4 LJ 60 5 LJ 62 13 LJ 62 14 LJ 62 15 C 1 44 1 1S48 1 1850 0 1851 1 1858 7 1858 10 1860 1 1562 16 aw PNG SVG Width 3024 Height 8000 It is recommended to export just one or two lipid series at once The chroms export can take some time depending on the data and the amount of selected hits The progress of the export is presented in a progress bar underneath the heat map Export PNG SVG Text Chrom export 38 50 1 34 7 2 show intern stand 2 v At the end of the chroms export the user is informed that the export is finished This picture is a chroms export of TG52 of the LDA It can be easily seen that analytes with more double bound elute slightly earlier This is a good quality criterion that the algorithm selected the correct hit Control elements e show intern stand should the internal standards be displayed in the heat map e show extern stand should the external standards be displayed in the heat map e isotopes what is the highest isotope number that should be used for the heat map for all of the analytes However the highest isotope number is determined for each analyte separately e g if 12 we have three samples 1 2 and 3 and for and 2 the isotopes 0 1 and 2 are found whereas in sample 3 just 0 and 1 are found just 0 and 1 are taken for the heat map double peaks should
6. 727939 822 75448 863 78103 10 48 2 51 94 5 0 0 0 802 705013 503 712259 820 73883 861 76538 11 48 3 51 82 5 0 800 589363 501 696639 818 72318 859 74973 12 48 4 51 90 6 D D D 798573713 799 580989 816 70753 857 73408 GE 50 0 53 102 6 0 0 QO 834 767613 835 7489 852 80143 893 82798 14 50 1 53 100 5 0 QO 832 751963 655 75924 850 78578 891 81233 15 50 2 53 98 5 D D D 830 736313 831 743589 848 77013 889 79668 15 50 3 53 96 6 0 0 QO 828 720663 529 727939 846 75448 887 78103 17 50 4 53 94 5 0 0 0 826 705013 627 712269 844 73883 885 76538 18 50 5 53 92 6 0 0 QO 824 589363 825 595539 842 72318 883 74973 19 50 6 53 90 6 D D D 822573713 823 580989 840 70753 881 73408 20 Ex I851 0 54 104 6 0 0 0 848 783263 649 79054 866 81708 907 84363 21 52 U 55 106 6 0 0 QO 862 798913 853 80519 880 83273 921 85928 22 52 1 55 104 5 D D 0 860 783263 861 9054 878 81708 919 84363 23 52 2 55 102 5 0 0 0 858 767613 85977489 876 80143 917 82798 24 52r 3 55 100 5 0 0 QO 856 751963 667 75924 874 78578 915 81233 25 52 4 55 98 5 D D QO 854 736313 855743589 872 77013 913 79668 26 52 5 55 96 6 0 0 0 852 720663 553 727939 870 75448 911 78103 27 52 B 55 94 5 0 0 QO 850 705013 851 712289 868 73883 909 76538 28 52 F 55 92 5 D D 0 848 689363 849 595539 866 72318 907 74973 29 62 8 55 90 6 0 0 0 846 673713 647 680989 864 70753 905 73408 30 54 O 57 110 5 0 0 0 890 830213 891 83749 908 86403 949 89058 3
7. Data Analyzer 1 6 0 FT settings Quantitation Batch Quantitation Statistical Analysis Display Results Settings License Help X Lipid Data Analyzer License Serial Number cq0037 Host ID 5273539 License Owner Demo License Type Single User License Module Licensed Expires Yes 2011 02 28 Re license Application X Lipid Data Analyzer Registration License String Name or Organisation 8 Help The help page contains links to several resources The first one is to an online help component LDA help explained in detail after the next figure Furthermore it provides links to the user manual this file the Examples pdf file that shows the functionality of the software with the aid of the published example data and the example data that can be downloaded from the Tranche repository The links to PDFs are provided to the home page and to local files that come with the installation package 18 m Lipid Data Analyzer 1 6 0 FT settings Quantitation Batch Quantitation Statistical Analysis Display Results Settings License Help The help component LDA Help To access the user manual click one of the links http iqenome tugraz atida 1 6 _DA 1 6 pdf Local instance in doc folder To access the examples document click one of the links http iqenome tugraz atida 1 6 Examples 1 6 pdf Local instance in examples folder The example data is available from Tranche
8. anal at not found this option tries to automatically quantify analytes which are grey at the retention time of the selected analyte whereby it does not take the theoretical isotopic distribution into account This procedure tries out all of the available quantitation methods starting with the 3D method then the greedy method followed by the MASPECTRAS and ASAPRatio method see chapter 4 3 For this option the application asks as well for which adducts modifications this operation should be performed like in Choose just one peak for doubles Mouse clicks on the sample group name e Left mouse button a bar chart for the sample group is displayed containing all the available analytes e Right mouse button just for sample Renaming of sample name Mouse clicks on the molecule name e Left mouse button a bar chart for the molecule is displayed containing all available samples groups e Right mouse button just valid for sample A popup menu appears containing 3 options 1 Remove analyte in all probes deletes one lipid in all result files 2 Select analyte If there are a lot of lipids and a lot of result files the removal of a lipid can be quite time consuming However if there are several analytes deleted it consumes the same time With this option analytes can be selected for the removal if selected the name of the analyte is surrounded by a blue rectangle The removal takes effect on all of the selected analytes
9. duplicate peak identifications be flagged with the yellow rectangle Settings button This is the major settings panel for the display value type relative value internal standard correction external standard correction consider dilution eiii Gil o value type which value should be displayed and used for the heat map or the bar chart relative value Just a value in arbitrary units This value could be the calculated quantity for the analyte itself or it could be standardized on standards depending on the other settings relative to base peak The values are calculated relative to the highest found peak of this lipid class relative to measured class amount Here percentual values are determined relative to the sum of all analytes for one sample standards are not considered in this sum of one lipid class relative to highest total peak The values are calculated relative to the highest found peak of all available lipid classes relative to total amount The values are calculated relative to the highest found peak of all available lipid classes amount end volume Returns the amount mol in the end volume before the MS measurement For this value standardization on an internal standard and absolute quantities see chapter 5 1 number 5 are required conc end volume Returns the concentration mol L in the end volume before the MS measurement For this value standardization on an internal standard and absolute quantities
10. in 2D or 3D mode In 2D mode the chromatogram is used and just the start and stop time has to be entered For the 3D mode the m z values have to be entered and an ellipse is fitted through these values 3D In the right menu of the chromatogram viewer the following options are available e Isotope Switches between the chromatograms of the different isotopes e Raw Smooth Toggles between the raw and the smoothed version of the chromatogram smoothed is the default one e t min t max and Zoom in This is for zooming the time axis With t min and t max the display range is defined and with Zoom in the settings are used for the viewer After zooming the and buttons can be used e Zoom all zooms the time axis out again 5 Statistics 5 1 File selection and settings The page for the selection of the files can be split into for 4 parts whereby changes in this section will just take effect in the statistics section after the Accept button had been pressed FA Lipid Data Analyzer 1 6 0 FT settings l b z nl x Quantitation Batch Quantitation Statistical Analysis Display Results Settings License Help Selection Add Files C Add Dir sb Remove all 3 Add to group file name directo 20100128 TAG 34 Massenliste TAG mitlIS xls FAMipidomicsi20100210 20100128 TAG 35 Massenliste TAG mitIS xls FAlipidormnicsi20100210 201001285 TAG 35 Massenliste TAG mit IS xls FMipidomicsi20100210 20100
11. next time somebody clicks on Save as default The settings file can be edited directly In the LipidDataAnalyzer properties file not only algorithmic parameters are stored but default settings for the display as well The changes take effect after the properties file had been stored and the LDA is restarted Path settings settings This part contains path settings to executables or required files e ReadWPath path to ReadW executable that is required if Thermo Finnegan RAW files are used directly e MassWolfPath path to the MassWolf executable if the Waters raw directories should be used directly e ElementConfig path to the required elementconfig xml file that contains the used chemical elements and the occurrence of their isotopes in nature Look amp feel settings settings e LookAndFeel This value is by default system which corresponds to your system specific look amp feel e g Windows If you prefer Java look amp feel change this value to java then you have the same look amp feel like in this manual Default quantitation settings properties This stores default settings for the quantitation input page e basePeakCutoff The default value for the Rel base peak cutoff see chapter 2 Excel result file settings settings e OverviewExcelWorkbook If true an overview tab is created in the Excel file 3D viewer default settings properties For the resolution of the 3D viewer
12. rectangle is around the peak If the mouse is hovered over one heat map cell a white rectangle is rendered and at the bottom of the application a line with the name of the lipid the name of the sample the value relative to the median a value depending on the Settings option can be standardized the original value of the quantitation and the amount of available isotopes is displayed Mouse clicks on cells in the heat map not valid for the group heat map e Left mouse button does not work for gray fields The LDA visualizes the quantitation see chapter 4 If the quantitation 1s based on several peaks due to adducts modifications the LDA jumps to the first one it finds 10 e Right mouse button does not work for gray fields an the ones with a yellow rectangle A popup menu appears with 2 options 1 Choose just one peak for doubles this option is for the automated removal of double peak identifications yellow rectangle It automatically selects in the other samples the one peak as correct that is closer to the retention time of the sample where the right mouse button has been clicked After this option has been selected the application asks for which adducts modifications this operation should performed x Are you sure you want to eliminate double peaks for 58 9 The peak nearest to the RT of 20100126 TAG 39 is taken v NH4 ACN v NH4 OK Cancel 2 Quant
13. standardization on an internal and or external standard and absolute quantities see chapter 5 1 number 5 are required relation to protein content This is for the standardization on a differently measured protein mass e g measured by a kit The values for the standardization have to be entered at the absolute quantities see chapter 5 1 number 5 If there is a standard available this value is presented in mol g else in AU g relation to neutral lipid content This is for the standardization on a differently measured lipid mass e g measured by a kit The values for the standardization have to be entered at the absolute quantities see chapter 5 1 number 5 If there is a standard available this value is presented in mol g else in AU g o internal standard correction 1s the standardization on an internal standard desired If yes it is possible to choose between 3 standardization methods most reliable standard Internally developed method that detects trustworthy standards and out of these a ratio to the other groups is calculated median The median of the standards of one sample is taken as reference value for standardization single standards several ones are available e g IS50 0 A standardization on every entered standard is possible o consider dilution Consider the dilution in the calculation o divisor unit If the value is standardized by some value this value defines the magnitude of the divisor e g pmol mL
14. 1 54 4 1 57 108 6 D D 0 888 814563 869 82184 906 84838 947 87493 32 54 1 2 57 106 6 D 0 0 886 798913 887 80519 904 83273 945 85928 Wn GREETE D ES 5 ACA c n n n n4 Faas nar Foor A I lt nna n47nn p naaeca r gt r The file consists of several sheets each representing a lipid molecule class If you want to analyze several lipid classes just make several sheets Second there must be a header column The header column must contain the following keywords otherwise it won t be accepted Name tR min at least one mass and at least one element column Except of the Name column which must be the first column the order of the columns does not matter for the Excel file Detailed description of the example in the figure columns from right to left e Name The name of the analyte e dbs This column is optional If the analyte name contains double bonds in it they should be entered in this column The values of this column must be integer format If the name of the analyte does not contain any double bonds just remove this column Generally it is possible to enter the double bonds in the Name column directly however the sorting of the analytes in the results is improved if this column is used e Element columns C H O P N D In this column the elemental composition chemical formula of the analyte has to be entered The values of this column must be in integer format If the
15. 128 TAG 37 Massenliste TAG mitlS xls jFMipidomicsi20100210 201001286 TAG 38 Massenliste TAG mitlS xls FMipidomicsi20100210 20100128 TAG 38 Massenliste TAG mitlS xls JF Mipidomics1201 00210 201001286 TAG 40 Massenliste TAG mitlS xls FMipidomicsi20100210 20100128 TAG 41_Massenliste TAG_mitIS xls JF Mipidomicsi20100210 201001286 TAG 42 Massenliste TAG mit IS xls FAMipidomicsi20100210 Quant file for correct analyte order E Data quantinputimasslist TG_with_IS xls Select gt normal eii Rename Remove X Delete Group eii Rename Remove XX Delete Group eii Rename Remove X Delete Group file name file name file name 20100128 TAG 34 Massenliste TAG mitlS xls 201001286 TAG 37 Massenliste TAG mitIS xls 201001286 TAG 40 Massenliste TAG mit IS xls 20100128 TAG 35 Massenliste TAG mitIS xls 20100128 TAG 38 Massenliste TAG mitIS xls 20100128 TAG 41 Massenliste TAG mitIS xls 20100125 TAG 35 Massenliste TAG mitlS xls 20100125 TAG 38 Massenliste TAG mitIS xls 201001285 TAG 42 Massenliste TAG mit IS xls Internal standard prefix S External standard prefix Ex I5 Load settings Remove abs settings use same settings for all experiments use same settings for all standards Dilution factor 66 67 Extvolume 20 w u L Extconc 1000 v u moin Intvolume 50 w u L Intconc 4 w u molt 34 35 36 37 38 39 40 41 42 Sample volume 2 vm L End volume 200 yi L Sam
16. 4 miz tolerance 0 02 exact mass 850 78578 Cancel OK 4 2 Visualization 3D viewer Stretch time 2 0 Stretch int 1 0 Stretchmiz _ 1 0 A Show Light Show Texture Selected C 2D Position Res 2 close t m z 0 001 Remres Add res Update The 3D viewer provides a menu bar at the top which can change the appearance of the viewer and a menu bar on the right side which is responsible for the resolution of the viewer The view on the plane can be changed when the mouse is clicked inside the drawing canvas Top menu bar e Stretch time int m z The 3D viewer itself treats every dimension equally so that every dimension should have the same length However for the chromatography it makes more sense to stretch the time axis Thus the stretch time 1s automatically set to 2 0 Any of the three dimensions can be stretched or shrunk with these three input fields The settings are applied after the Update button has been pressed e Show Light The 3D viewer has a light source falling on the plane resulting in bright or shadowy spots The light effects can be switched off with this check box e Show Texture In order to give the picture more contrast a texture is rendered over the plane The texture can be turned off with this check box e Selected If selected the quantified and stored peaks are colored in read the ones that are just quantified and not stored are colored in green If not selected th
17. If elements are lost by the modification enter a in front e g NH4 would correspond to a loss in ammonium A space is interpreted as that means if you enter N H4 this would correspond to N H4 Additionally the charge state can be entered by charge charge_state If this information is missing in the brackets a charge state of 1 is assumed 21 e tR min This is the retention time column The input of the retention time is optional If it has not been entered the whole chromatogram 1s searched for a peak if the checkbox Find molecules where retention time is unknown is checked Third it is possible to enter a general start and stop retention time for a lipid class The notation is Start RT and Stop RT These cells can be anywhere before the row with the header row Appendix B Configuration settings This chapter covers the manual adaption of LDA setting parameters General settings can be found in the settings file and machine specific ones in the LipidDataAnalyzer properties file For each machine such a properties file is present in the propterties folder in the installation directory It is recommended to change the settings for the machine in the corresponding file in the properties folder and after saving this file to select the machine and click on Save as default see chapter 6 If the settings are changed directly in the LipidDataAnalyzer properties these settings are overwritten the
18. L the intermediate mzXML is deleted automatically after conversion to chrom format Then on the chrom files the quantitation is started The results of the quantitation will be stored in an Excel file whereby the name of the results file will be path of the chrom file without suffix quant file name e g raw file F lip domics 20100210 20100126_TAG 34 chrom quant file FMipidomics TG NHA4 ACN xls result file F lipidomics 20100210 20100126_TAG 34_TG_NH4_ACN xls 3 Batch quantitation The quantitation pages for Quantitation and Batch Quantitation look similar The difference is that for Quantitation the file chooser requires a single file whereas in Batch Quantitation a directory containing the files to quantify and a directory containing the Excel mass list files 1s required FA Lipid Data Analyzer 1 6 0 FT settings Quantitation Batch Quantitation Statistical Analysis Display Results Settings License Help Raw files Quant files Time before tol 2 min Time after tol al min Rel base peak cutoff 04 RT shift ool min Isotopic quantitation of a isotopes where 4 isotopic peak s have to match Find molecules where retention time is unknown Processors for quantitation 1 Start Quantitation In contrast to the normal quantitation in batch mode a list will appear that shows the progress of the quantitation The files receive a green check mark if t
19. Lipid Data Analyzer 1 6 User Manual OeneraboVervie W ae ea Neu dem d a ec Oda NM edad Made du MM ae utere i gt AON OT Em gt JBalclbquaBtt UO car ee en cre Du MEDLINE uae UK EPIS EDI CILE 3 4 Manual verification visualization ccccccsssessssseeccccceneeesssceeccccccseeesseseesccessueeesseseesees 4 Al NiSUaliZalOmie Tere IMC iUne 4 42 WMasualizatton 3D VIeWeE 2 2 5 4 3 Visualization Chromatogram viewer esses eene nennen eene 6 SMEs MEN 7 Sl Fileselecuon and setine S iu pce n edicta nodu made 7 5 2 Heat map and visualization settings ccccccccccssssseseeeccccceeeaeeeseeeeeeceeesaeaeseeeeeeeeeeaaas 9 I DUC sonst een 14 Ser Overview Dar Chan were UU 16 MEE cin See RR 17 T C Ic M 17 cra er C X 18 Appendix A Preparation of the mass list Excel file essen 21 Appendix B Configuration Settings o oie quebec u Mat 22 Pati Setn 22 Irae Eua Er lin rennen PE 22 Peuh quanttCitiOD Se LEONES caseo nee Dee alien 22 Peer sul Tlesetinss a oer dta etate tesa nile ea 22 SD viewerdeFault Settings et ea emo tes utu Asse 22 Standards Prex SEUNG nenn tesi ted ipao M DD QU EE Ub rope pi pabus 22 MAME CMO Settings anne e 23 General algorithm Sti SS russian een 23 Memory SILIO S uicit eodd Dex qium een 23 JADpendix Ge 17 Fabsellinss naar Beten 23 MS Instrument specie Se
20. LipidDataAnalyzer properties file see Appendix B Configuration settings FA Lipid Data Analyzer 1 6 0 FT settings i l 10 x Quantitation Batch Quantitation Statistical Analysis Display Results Settings License Help Raw file Quantitation Time before tol 2 min Time after tol 3 min Rel base peak cutoff 0 1 Xo RT shift 0 0 min Isotopic quantitation of a isotopes where al isotopic peak s have to match Find molecules where retention time is unknown Processors for quantitation 1 Start Quantitation Raw file File in mzXML or chrom format is required However it is possible to use Thermo RAW format and the Waters Mass Lynx raw directories directly if XCalibur or MassLynx 1s installed Quant files Here the name of an Excel file has to be entered containing the mass list The format of the file is described in Appendix A Preparation of the mass list Excel file Time before tolerance This field specifies the retention time tolerance in minutes before the entered retention time in the Excel file Example is given after Time after tolerance This field is just used if a retention time has been entered in the mass list Excel file Time after tolerance This field specifies the retention time tolerance in minutes after the entered retention time in the Excel file Example is given in the next paragraph This field is just used if a retention time has been enter
21. NH4 A 1 12894216E8 503 NH4 5 8722547E8 50 4 NH4 A 2 644618E7 50 4 NH4 1 3079372E8 150 5_NH4 A 6363079 0 50 5 NH4 2 274767E7 50 6 NH4 A 1600180 2 50 6 NH4 4030983 0 IEx 1851 0 N 1 9128E8 52 0_NH4 A 1 3580512E 52 0 NH4 4 5505308E T 52 1 NH4 A 2 095768E8 521 NH4 52 2 NH4 A 5 3 52 3 NH4 A 7 1553485E8 52 3 NH4 4 28250304E9 52 4 NH4 A 2 51418832E8 52 4 NH4 1 42083187E9 52 5 NH4 A 6 2537272E7 52 5 NH4 3 90111456E8 52 6_NH4_ 52 7 _NH4 A 52 7 NH4 52 8 NH4 258178 44 arb units AU CTroz 848 7701 54 0 NH4 A 46983240 A 7 24568 22 9 retis Isotope 0 54 0 NH4 7206967 0 deris e C Raw om 541 NH4 A 1 48712432E8 54 1 NH4 5 3742566E8 54 2 NH4 A 2 44870784E8 54 2 NH4 54 3 NH4 54 3_NH4 A 1 59441894E9 1 28420984E8 1 2609696E9 LEA A ILIA us 4 2nn4 2 2rol Y v 15000000 10000000 5000000 Gain Gain sa gt gt eee Smooth mz t min t m ax Zoom in Zoom all Save Time min v Show 2D Vie 10 20 30 The menu has at the top a selection box where you can switch between the different lipid classes Then a table follows whereupon the name of the analyte is in the first column and the area in the second The display nam
22. as soon as the Choose just one peak for doubles option is pressed Here again a confirmation box appears asking for the adducts modification on which the removal should be performed Export bar The picture of the heat map or the data respectively can be exported in the following formats PNG portable network graphics SVG scalable vector graphics Excel Microsoft Excel Text text based format tab delimited Excel and Text export the values in the type that are selected by the user see next paragraph Control elements Furthermore there is the option to export results in mzTab format in order to submit them to a public repository For mzTab the raw peak areas are exported these can be affected by standardization and isotope setting only The mzTab button exports all lipid classes at once and generates a file containing all experiments the data can be exported to chromatograms with the Chroms button Here the exported analytes experiments and modifications can be selected 11 Analyte Analytes 46 0 _ 48 0 I O 48 1 48 2 48 4 v 50 1 a 50 2 e 50 4 v 50 5 v 50 6 Ex 1551 0 52 0 lu 5271 52 2 52 3 52 4 L 52 5 52 6 LJ 52 7 L 54 10 56 10 58 8 LJ 56 0 _ 56 1 LJ 56 2 LJ 56 3 56 4 O 56 5 LJ 56 6 56 7 8 56 8 LJ 56 9 LJ 56 11 58 9 LJ 56 12 58 10 58 0 LJ 58 1 LJ 58 2 LJ 58 3 C 58 4 LJ 58
23. default settings can be entered e threeDViewerDefaultTimeResolution The default time resolution of the viewer e threeDViewerDefaultMZResolution The default m z resolution of the viewer Standards prefix settings settings The field for the prefixes of the standard can be set by default see chapter 5 1 e SDefaultInput Default prefix for the internal standard e ESDefaultInput Default prefix for the external standard 22 mzXML chrom settings properties These are settings for the translation from the mzXML to the chrom format e maxHileSizeForChromTranslationAtOnce An approximate value how many MB can be read at once into the memory The mzXML is translated into the chrom in several rounds If there is e g a mzXML file with 200MB and an m z range from 600 1400 then this file will be translated in two rounds if this parameter is 100 first round masses from 600 1000 second round masses from 1000 1400 Thus this parameter does not directly correspond to the required memory the required memory is normally more If this parameter would have been 200 the whole file would have been translated in one round The reason for this parameter is to permit the translation of huge files on machines with low RAM If this parameter is increased the translation time is decreased If this parameter is too high a java lang OutOfMemoryException will be thrown and the file cannot be translated e chromMultiplicationFactorForInt and chromMultip
24. e chrom file and the results Excel file FA Lipid Data Analyzer 1 6 0 FT settings B x Quantitation Batch Quantitation Statistical Analysis Display Results Settings License Help ipidomics 201 002101201001 26_TAG 34 chrom Open Chrom lomicsi20100210120100126 TAG 34 TG NH4 ACNxISD Open Result After the files has been selected press Start Display The now appearing display can be divided into two parts 1 a menu on the left side see 4 1 2 a display frame whereupon the upper part shows a 3D view see 4 2 and the lower part displays the 2D chromatogram see 4 3 4 1 Visualization left menu FA Lipid Data Analyzer 1 6 0 FT settings E zii x Quantitation Batch Quantitation Statistical Analysis Display Results Settings License Help FF Mipid omicsi20100210120100126 TAG 34 chrom Open Chrom Start Display F Aipidomicsi20100210120100126 TAG 34 TG NH4 A Open Result Stretch time 2 0 Stretch int 1 0 Stretch mz 1 0 Show Light Show Texture Selected C 2D Position Update Name 48 0 NH4 48 1 NH4 A 1 8613 48 1 NH4 1 02946936E8 48 2 NH4 1 03754016E8 48 3 NH4 A 1 0168225E7 48 3 NH4 2 9692476E7 48 4 NH4 89298237 0 50 0_NH4 6184432E7 50 1 _NH4 A 1 85691296E8 50 1 NH4 9 1039354E8 50 2_NH4 A 2 49829344E8 50 2_NH4 1 2182569E9 50 3
25. e coloring depends on the signal intensity green low intensity yellow medium intensity red high intensity e 2D Position This shows the stripe that has been taken to extract the chromatogram in gold Right menu bar e t Is the time resolution in seconds e m z Is the m z resolution in Daltons e Add res The viewer has the ability to change the resolution level depending on how close the view is on the plane Several resolution levels can be entered If an additional resolution level is required just press this button The point where the resolution is changed is defined by the d field e d Isthe distance field it appears if several resolution levels are set The distance is always measured from the center of the plane 1 corresponds to a total distance of the m z axis to the center of the plane This value is rather empirical but due to general applicability to different resolutions this approach had been chosen Rem res Removes a resolution level e Update This button has to be pressed when the resolution settings should take effect for the 3D view Mouse buttons e Left mouse button Changes the viewing angle on the canvas e Middle mouse button Zooming in and out e Right mouse button Moving the plane 4 3 Visualization Chromatogram viewer Aub Units AU A T 24588 22 93 m a Isotope lo C Rau m2 Int 7 45585 smoot EE 15000000 Gain t min t m ax 10000000 Gain Zoom in
26. e consists in principle of name double bonds e g 50 2 If there 4 exists more than one adduct modification the modification name is added with modification name e g 50 2 NH4 At the bottom of the menu the range for the 3D viewer can be set The current center m z value of the extraction 1s displayed in the 2D viewer see chapter 4 3 After the m z range has been changed the Update button has to be pressed to change the display The Show 2D View select box can fade out the 2D viewer If a row of the table is clicked the peaks are displayed in the 3D and the chromatogram viewer If the row is clicked with the right mouse button a popup appears Ex I851 0 1 9128E8 52 A4RRTRA F7 This allows to add an additional analyte or to delete the selected one To delete several analytes at once keep Ctrl or Shift pressed and select the analytes this will prevent refreshing of the 3D viewer and save time As soon as you release Ctrl or Shift the 3D viewer will be refreshed by the analyte that was selected at last Before after just determines the position where the analyte will be added The adding of a new analyte requires the name of the analyte its chemical formula the name and the formula of the modification if any the m z tolerance of the chromatogram and the exact mass x Add a new analyte before 50 2 Name 5 0 1 Formula C53H10006 modification INH4 Formula N H
27. ected it is assumed that the standards are added to each experiment at the same amounts So a specific setting for each experiment is possible e use same settings for all standards If this checkbox is selected it is assumed that all of the external standards Ext volume and Ext conc has to be entered and internal standards Int volume and Int conc has to be entered are added at the same amounts If not a list of the standard appears and each standard can be entered separately The dilution factor corresponds to the dilution from the adding of the external standard until the internal standards are added If the settings are entered for each experiment separately the buttons Apply to all and Apply to group appear These buttons permit the propagation of the entered values to all experiments or to the members of a group The lower box is for the specification of sample specific settings like sample volume before sample preparation and end volume after sample preparation The sample volume and the end volume are mandatory whereas the Sample weight the Protein conc and the Neutral lipid conc are not If latter values are entered standardization on these values 1s possible The Apply to all and Apply to group buttons permit the propagation of the entered values to all experiments or to the members of a group 5 2 Heat map and visualization settings After the Accept button had been pr
28. ected molecules in the mass list without retention time entry are quantified discarded otherwise However the quantitation of molecules without retention time consumes more time Processors for quantitation The amount of processors that shall be used for the quantitation LDA detects the amount of processors available automatically The default value is always n 1 and the software does not allow for more than n processors Start Quantitation This button starts the quantitation FA Lipid Data Analyzer 1 6 0 FT settings u B x Quantitation Batch Quantitation Statistical Analysis Display Results Settings License Help Raw file F Mipidomicsi20 100210120100126_TAG 34 RAW Select Quantitation ILALDA PapenquantExcelFilesuwnasslist TG with IS xls E Select Time before tol 2 min Time after tol 3 min Rel base peak cutoff 0 0 RT shift 0 0 min v Isotopic quantitation of 2 isotopes where 1 isotopic peak s have to match Find molecules where retention time is unknown Processors for quantitation 1 Quantifying TG 48 2 3 101 Bye After the quantitation has been started first Thermo RAW format and the Waters Mass Lynx raw directories are translated in mzXML if the quantitation is based on the raw file formats Second mzXML files are translated in the internal chrom file format If the raw file is Thermo RAW or Waters 2 Mass Lynx and not mzXM
29. ed an overview is displayed containing the standards used If for one lipid class no standard is available the class is neglected like here LyPC Without the consider standard these classes would have been displayed Attention without the consider standard it is assumed that all classes ionize equally As quant type just area absolute and percentual values 1s available 16 6 Settings The LDA can be used for several MS machines The Settings tab has been introduced to change these settings easily The settings must not be changed while a calculation is running FA Lipid Data Analyzer 1 6 0 FT settings l Bl x Quantitation Batch Quantitation Statistical Analysis Display Results Settings License Help Please do not change the settings while a calculation is running MS settings FT Save as default The current settings are displayed in the title of the application The Apply button applies the settings to the current session The Save as default button sets the selected settings as default which will loaded automatically at the next start of the LDA 7 Licensing In order to view the license the tab License has to be clicked Then a dialog window shows the details about the current license 1 in next figure If you acquired a new license click on Re license and a dialog window appears where the license hash plus the name of the institution has to be entered 17 Lipid
30. ed in the mass list Excel file Example for time tolerance setting E g in the excel file the retention time for a specific analyte 1s specified with 24 minutes in the time before tolerance 2min and in the time after tolerance 3min is entered Then the algorithm will look for the analyte in the time range from 22 27 minutes Rel base peak cutoff Peaks that are less intense than this per mille value of the highest identified are discarded by the algorithm For the determination of the highest peak just found analytes are taken into account irrespective of the analyte class This threshold can accelerate the quantitation since intensities that are too small are discarded before the 3D quantitation 1s performed RT shift The retention time could shift from batch to batch The entered value will be added to the retention time defined in the mass list file Thus the mass list file has not to be changed every time Isotopic quantitation of isotopes where isotopic peaks have to match The checkbox indicates if in addition to the 0 peak other isotopic peaks should be quantified The first value determines the amount of additional isotopes to be quantified e g 2 would mean that 0 1 and 2 isotopes will be quantified The second value determines how many isotopes have to conform the theoretical isotopic distribution e g 1 just the 1 peak has to conform Find molecules where the retention time is unknown If this checkbox is sel
31. essed a tab for each lipid class appears This tab again contains a tabbed pane with the entries Heatmap and Bar chart and if groups are selected Group Heatmap and Group bar chart The Heatmap tab is selected by default The tab itself contains the heat map at the top followed by an export bar and some control elements at the bottom J Lipid Data Analyzer 1 6 0 FT settings 2 2 B x Quantitation Batch Quantitation Statistical Analysis Display Results Settings License Help Selection TAG Heatmap Bar chart Group Heatmap Group bar chart Export PNG SVG Excel mzTab Text Chr vr show intern stand 2 w isotopes v show extern stand double peaks Settings Select molecules Combined chart Export options 4 Il Li Heat map At the top the heat map contains a color legend The values in the map are calculated relative to the median of one molecule over all samples groups If the intensity of a molecule of one sample group is lower than the median it s colored green the ones that are higher are red and the ones around the median are black If one analyte could not be quantified it 1s gray In the heat map the samples groups are organized horizontally and the lipids vertically If a quantitation is based on more than one peak could be an ambiguous identification each isotope separately a yellow
32. esults Each group is displayed in a small table and the following buttons are available e Rename Renames the group e Remove Removes members of one group after they had been selected in the table where the group members are shown e Delete Group Deletes the entire group Number 4 is for the selection of molecules as standard It is assumed that the standards in the results carry a certain prefix e g IS or Ex IS This prefix has to be entered in the two input fields The usage of standards is not mandatory so these fields can remain empty If there are prefixes entered and no molecule carries these prefix the standardization is neglected Number 5 is for the standardization on certain values like the protein content or the calculation of absolute values just valid if there are standards and the ionization efficiencies of the analyte can be assumed to be similar like the one of the standards This section is not mandatory Before this section can be used the results have to be selected first in number 1 and the prefixes for the standards have to be defined in number 4 Then the Add absolute settings button has to be pressed The application reads now the result files and looks for standards The upper box of this section is for the assessment of absolute values for the standard The box has a tab for each lipid class so the settings can be made specifically for each class e use same settings for all experiments If this checkbox is sel
33. hey are quantified successfully If not a red X B Lipid Data Analyzer 1 6 0 FT settings 4 m x Quantitation Batch Quantitation Statistical Analysis Display Results Raw files FipidarmicsiWikii201 00901 Eichkurven 20100827 Bvy Quant files FlipidomicsiMikiimassList Time before tol 2 min Time after tol 3 min Rel base peak cutoff 0 1 RT shift 0 0 min Isotopic quantitation of 2 isotopes where 1 isotopic peak s have to match Find molecules where retention time is unknown Processors for quantitation 1 Start Quantitation Data iauant File Progress 20100827 wvB eichreihe 4a chram 20100831 Massenliste EICHKLRVE xls Finished 20100827 WB eichreihe Tb chrom 20100831 Massenliste EICHKURVExIS 20100827 WB eichreihe l2c chrom 20100831 Massenliste EICHKURVExIS 20100827 WB eichreihe Ta chrom 20100831 Massenliste EICHKURVExIS 20100827 WB eichreihe 12b chrom 20100831 Massenliste EICHKURVExIS 20100827 WB eichreihe 12a chrom 20100831 Massenliste EICHKURVExIS 20100827 WB eichreihe 3c chrom 20100831 Massenliste EICHKURVExIS 20100827 WB eichreihe 3b chrom 20100831 Massenliste EICHKURVExIS 20100827 WB eichreihe 3a chrom 20100831 Massenliste EICHKURVExIS S 20100827 WB eichreihe Bc chrom 20100831 Massenliste EICHKURVExIS 4 Manual verification visualization In order to visualize the results 2 files are required th
34. licationFactorForInt This parameter define the highest resolution of the chrom file e g chromMultiplicationFactorForInt 1000 and chromMultiplicationFactorForInt 1 would mean a max resolution of 0 001Da chromMultiplicationFactorForInt 1000 and chromMultiplicationFactorForInt 5 would mean a max resolution of 0 005Da chromMultiplicationFactorForInt 100 and chromMultiplicationFactorForIntz1 would mean a max resolution of 0 01Da General algorithm settings properties This section contains the most general settings of the algorithm A detailed description of the parameters of the 3D algorithm is beyond the scope of a user manual The parameters are described in the LipidDataAnalyzer properties file e coarseChromMzTolerance the m z range that is used for the extraction of the first chromatogram of the algorithm which is displayed in the 2D viewer see chapter 4 3 e chromSmoothRange the smooth range in seconds for Savitzky Golay filter of the first chromatogram e chromSmoothRepeats the smooth repeats for Savitzky Golay filter of the first chromatogram e use3D If yes the 3D algorithm is used by default for the quantitation else the 2D algorithm Memory settings bat sh or vmoptions The memory settings for the executables can be changed in the Lipid Data Analyzer vmoptions and for the console in the bat or sh respectively The Xmx parameter corresponds to the maximally reservable memory and Xms the immediately reserved memo
35. ment specific settings The exception is the contact settings which must contain at least two commas The first comma separates the name the second one the email address and the rest of the commas are included in the affiliation The contacts have to start with one and MUST have consecutive numbers e g contact 1 name first contact Semail first contact S affiliation first contact contact 2 name second contact email second contact Saffiliation second contact Other supported metadata settings of this file are e species from which species originates the sample e tissue from which tissue originates the sample e celltype the cell type of the sample If any of the values are unknown or cannot be assigned they should be commented or left out The export to mzTab is possible with missing values Appendix D Trouble shooting It there are any errors please start the LDA with ux console x C WINDOWS system32 cmd exe 10 x D Deve lopment LipidDatafnalyze rr iyjava jdkt 6 8_BS bin java Xns256n aoc Quantitation 3E Batch Quantitation Statistical Analysis Display Results Settings License Help opiti Es ra risen de eee Open Chrom elopment LipidDatafnalyzer LipidDatafnalyze roperties r j Start Display Then please try to reproduce the error Afterwards please make a copy of the console output and send it with a detailed description of the preceding user interactions to the developers
36. mne a sn ak etd ee ia dites Dudas 23 Other qmm T aD Se Ina ae ee euere 24 Appendix D Trouble shootins una u eum Fut Rar e aM p epe Ud ES 24 1 General overview There are two ways to start the program one as plain executable and one with a console If the console is used a command line window black one appears which is the output of the program log FA Lipid Data Analyzer 1 6 0 FT settings BE Bl x Quantitation Batch Quantitation Statistical Analysis Display Results Settings License Help Raw file Quantitation Time before tol I 2 min Time after tol 3 min Rel base peak cutoff 04 RT shift oo min Isotopic quantitation of 2 isotopes where 4 isotopic peak s have to match Find molecules where retention time is unknown Processors for quantitation 1 Start Quantitation In the LDA window six tabs are available For Quantitation and Batch Quantitation see chapter 2 and chapter 3 the Quantitation and Batch quantitation section for Results Analysis see chapter 5 the Statistics section for Display Results see chapter 4 Manual verification visualization for licensing visit chapter 6 and for help chapter 8 2 Quantitation In order to start a single quantitation a raw file Raw file and an Excel file Quantitation containing the mass list is required The default settings for the input fields can be entered in the
37. nse Licensing Help Help Furthermore the following additional help topics are provided Preparation of the mass list Excel files Configuration settings Trouble shooting The help component contains 4 parts 1 Information panel It contains information about the current help topic Cross links to other help topics are inside these pages 2 Tabs containing different ways to search for the desired help topic e Table of contents The topics are organized like in a table of contents in a document e Index The help topics are ordered alphabetically e Search Enables to search the help for words 19 e Favorites Organization of favorite pages of the help The user has to right click with the mouse in the tabbed pane of favorites and can add help pages to this menu 3 Navigation tree for the different tabs explained before 4 Control bar This bar contains buttons for previous page next page home reload page print and print page setup 20 Appendix A Preparation of the mass list Excel file The Excel file containing the mass lists must follow some conventions E TG NH4 ACN xls Ac B c Jerez ede ii e L M N SIE lix 1 zi 25 3 3 4 TG C H 0 P N D M 5 2 9 14 6 0 0 218 6 Z Name dbs C H 0 P N D M MH mass form NH4 name NH4 mass form N2C2H7 name NH4 ACN tR min 8 a I gg I I T 806736 SU 733583 974 7 7U 865 7 9660 9 48 1 51 96 6 D 0 0 804 720663 505
38. or our homepage Link to Tranche download Link to Genome download Please consult the examples document chapter to avoid unnecessary downloads After the link LDA help had been clicked a window containing the help component pops up Lipid Data Analyzer Help Table OrContents index Search Favorites 7 E General overview 3 Overview 3 Quantitation 3 Batch Quantitation C Statistical Analysis gt C Display Results 3 Settings 5 Licensing 3 Help 3 Preparation ofthe mass list Excel files 3 Configuration settingss 3 Trouble shooting 3 There are two ways to start the program one as plain executable and one with a console If the console is used a command line window black one appears which is the output of the program log A ipid Data Analyzer 1 5 1 Quantitation Batch Quantitation Statistical Analysis Display Results License Help J Rave file ll seed Quantitation Select Time before toL 2 min Time amer toL 3 min ReLbase peakcutom 0 Xe RT Shift w Isotopic quantitation of 2 isotopes where isotopic peak s have to match Find molecules where retention time is unknown Start Quantitation In the LDA window six tabs are available Quantitation Quantitation f Batch Quantitation Batch quantitation Statistical Analysis Statistical Analysis Display Results manual inspection Display Results Settings Settings Lice
39. or respectively groups are displayed 5 Color selection In this legend the used color and the corresponding item is displayed If the mouse is clicked on this legend the color can be changed 6 Export bar The picture of the heat map or the data respectively can be exported in the following formats PNG portable network graphics SVG scalable vector graphics Excel Microsoft Excel Text text based format tab delimited 5 4 Overview bar chart If there are several lipid classes available a bar chart for the overview of the contribution of the separate lipid classes is available FA Lipid Data Analyzer 1 6 0 FT settings ni x Quantitation Batch Quantitation Statistical Analysis Selection SM PE PC LyPC DG PS Overview O view Group quant type percentual values M single sided double sided logarithmic linear 1 v isotopes amp standard deviation 1 0 2 standard error mean License Display Results Settings _ export deviation value analyte in column exp in column Overview relative to total amount of classes percent 4 1 0 SD T gt Ld e S i El Group 1 u Group 2 E Group 3 E Group 4 Export PNG SVG Excel Text consider standard consider dilution Accept Selected This overview has the option to consider standard the current standard settings are taken and to consider dilution If the consider standard is select
40. ple weight 800 v m g Protein conc 178 8 w m gl Neutral lipid con 58 64 w m g L Number is for the selection of files e Add Files This button allows selecting result files Excel for the analysis The files appear then in the table below e Add Dir Does the same like Add files but reads automatically the content of a whole directory and adds all of the Excel files in the directory to the table e Remove Removes one ore several results from the table The files have to be selected before in the table e Remove all Removes all files in the table e Add to group Is for the grouping of results First a set of files has to be selected in the table and then this button has to be pressed Then a dialog box asks for the name of the group and the group is added to number 3 the grouping part Number 2 is for the sorting of the analytes If in the quantitation step several analytes cannot be detected not all of the analytes appear in the Excel results file Then it is hard for the algorithm to specify the correct order At number 2 the original quantitation file can be provided The analytes will then be displayed in the order they occur in the Excel file The provision of the original Excel file is not mandatory but can be helpful if several analytes were not found Number 3 displays the groups they have to be selected at number 1 The grouping is not mandatory but it can help in the interpretation of the r
41. re is a value in float format e g the M column the column will be ignored as elemental column a warning at the beginning of the quantitation appears A column is an elemental column if it contains just one or two characters beginning in upper case and ending lower case e g H Na He P etc If you require new elements just add an additional column and enter the probable occurrences in nature in the elementconfig xml file see Appendix B Configuration settings where to find this file e Additional calculation columns e g M MH These columns are not read by the application and are just a help for the user to calculate the masses Do not mind that the comma in the float value is a and not a the Excel I used for the screen shot is using the German notation e Mass columns mass A mass column contains the mass values to be quantified There can be several mass columns because of different modifications adducts A mass column must contain the keyword mass followed by round brackets Within the round brackets there must be the keywords name and form followed by square brackets In the square brackets after name the display name has to be entered after form the chemical formula The chemical formula of the modification adduct must be in the notation element_symbol element_amount e g H4 If no element amount is entered it is assumed to be one e g NH4 N1H4
42. re none m u n p f a and o e radio button single sided double sided just enabled for area relative to molecule and area relative to standard For single sided the values always start with 0 For double sided the area is relative to the median standard and lower values are painted in negative direction and higher values in positive direction e radio button logarithmic linear Available for area absolute percentual values and absolute quantity This option switches between linear and logarithmic log10 display e radio button log10 log2 Available for area relative to molecule and area relative to standard Toggles between decadic and dual logarithm e isotopes Determines the amount of isotopes that should be used for the quantitative value 15 2 Deviation settings Available just for groups This value defines how the error bars should be displayed The deviation can be displayed at any multiplicative value 1 0 s default and the standard error mean can be displayed 3 Export settings Settings for the export in Excel and Text format These are the same like in chapter 5 2 Export options 4 Barchar The main painting area At the top in the center the lipid class and the value type is displayed On the left written vertically the used standardization procedure is displayed At the top of the vertical axis the current unit is displayed In the horizontal axis the molecule samples
43. ry The Xms value should be approximately half of the Xmx value Appendix C mzTab settings LDA provides the possibility to export metadata in the mzTab format None of this information is mandatory for the mzTab export but helps for the reproducibility of your data All changes in these settings require a restart of the LDA application The metadata is split in two configuration files 1 properties file containing information about the mass spectrometer ii mzTab properties containing experiment specific information such as affiliations and sample origin information MS instrument specific settings properties In the current implementation the following metadata can be exported in mzTab format mzTabInstrumentName the name of the instrumentation mzTabInstrumentlonsource the ion source of the instrumentation e g ESI or MALDI mzTabInstrumentAnalyzer the analyzer of the instrument e g ion trap FT ICR etc mzTabInstrumentDetector the ion detector e g electron multiplier 23 Examples are provided within the files Be sure for all parameters that the entries contain always three otherwise this setting will be neglected If you are using as recommended controlled vocabulary please crosscheck its validity http www ebi ac uk ontology lookup Other mzTab settings mzTab properties All the other metadata for mzTab is stored in this file The entries here must contain three commas such as in the MS instru
44. ue analyte in column exp in column m export retention time z m D c m t 5 2 t o E u e w B m c m t E 5 2 t E Ui a 5 a N z m D c m t high fat normal fasted 9 Export PNG SVG Excel Text 6 The parts encircled in violet are just visible for the grouped view In general the bar chart painter can be separated into 6 parts Display settings This 1s for changing the bar chart display e quant type Determines the type of values that have to be displayed a area absolute The absolute area values as they are returned from the algorithm or if there is a standardization on an internal or external standard these values are the standardized ones b percentual values The selected molecules group sample correspond to 100 and the values are displayed relative to this amount c arearelative to molecule The values are displayed relative to the median d arearelative to standard The values are displayed in relation to the calculated standard just available if a standardization 1s selected e absolute quantity The values are displayed in the quantity that are selected at 5 2 Settings button The bar chart will always appear in this setting by default except if relative value 1s selected in the settings e a small select box after the quant type This box is just visible for the absolute quantity settings The input specifies the magnifier available a
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