Addendum to DeCyder 2D™ 7.0 User Manual, 28-9414
Contents
1. i th protein V gt is the spot volume in the other sample for the i th protein Repike IS the raw mean volume ratio for all spike proteins Spike normalization in BVA module Inthe BVA module spike normalization will normalize the standard abundance value for all proteins so the mean value of all spike proteins standard abundance will be set to 1 0 The spike standard abundance is calculated as follows S Via Vinel Sspike Where S is the spiked standard abundance for the i th protein Vix is the spot volume for the i th protein and the x th spot map Vigs Is the spot volume for the standard spot corresponding to the i th protein and the x th spot map Sspike IS the mean standard volume ratio for all spike proteins 2 Addendum to DeCyder 2D User Manual 28 9680 43 AA Addendum to DeCyder 2D 7 0 User Manual 28 9414 47 AA Details of new features in DIA module Details of new features in DIA module 28 9680 43 AA Addendum to DeCyder 2D User Manual In the DIA Main window there are two visible changes in version 7 1 e inthe Spot Control Panel there is a new checkbox Spike e in the table view there is a new column Spike The word Spike in the new column indicates that the protein is assigned as a spike protein m lel wih iih ta FMS DFE mE LA TEET p zeafzs When spike normalization will be used in the BVA module perform all normalization in the BVA module Spike normalization performed in the DIA module w2. Addendum to DeCyder 2D 7 0 User Manual 28 9414 47 AA Introduction DeCyder 2D version 7 1 is an upgrade from 7 0 with a new functionality to normalize abundance values using spike proteins This is a complement to the present model based normalization It is suitable for experiments where one or several proteins have the same presence in all samples and can be used as references for the normalization The spike normalization is added in the DIA and the BVA modules Spike Normalization Limitations of model based spot normalization The model based spot normalization in DeCyder 2D DIA and BVA is based on the assumption that the majority of all proteins in a gel keep their expression level between the different samples in an experiment In a classic DIGE experiment it is important that at least 100 spots preferably 2000 3000 spots are detected to get a valid normalization However this approach is not optimal for applications with samples that have few protein spots or samples where the majority of the proteins differ in expression level between samples Summary and benefits of spike normalization To expand the usability of the software an alternative normalization tool based on so called spiked proteins is introduced in DeCyder 2D 7 1 With this it is possible to normalize the proteins in an experiment to user selected proteins in the samples Spike proteins must be added to the samples prior to the CyDye labelling step It is importa
3. When spike normalization will be used in the BVA module perform all normalization in the BVA module Spike normalization performed in the DIA module will not be included when importing the workspace into the BVA module How to use spike normalization in BVA 1 Identify the spiked protein s using the gel view in P Protein Mode Check the appearance number for all spiked proteins For a good normalization all spiked proteins should be present in all spot maps If a spike protein is mis matched or missing in a spot map go to M Match Mode Use the spot editing options in the Data View Controls to correct the matching until all spike proteins are present and matched in all spot maps See 5 9 2 Available editing options in DeCyder 2D User Manual 28 9680 43 AA Addendum to DeCyder 2D User Manual 5 Addendum to DeCyder 2D 7 0 User Manual 28 9414 47 AA Details of new features in BVA module 2 Goto P and select the spiked proteins one by one and tick the Spike checkbox to indicate that it is a spiked protein Protein ID Protein AC Name Comment pl erm en L1 List Change list IV Spike Mw Daf POI Pick spot map Confirm Tip Use the comment field for the spiked protein name to have better control and be able to vary the assigned spiked proteins 3 When all spiked proteins are assigned click on the Process menu and select Normalize on Spiked Proteins to perform the spike normalization 4 Verify the normalization by viewing the
4. e DeCyder database and then uninstall the database and the program part see Section 5 Uninstallation in the DeCyderlnstallationGuide pdf before installing The Oracle Server can remain installed Licensing Access to the spike normalization is controlled by the eLicense system For details on eLicensing see Section 4 eLicense installation and activation in the DeCyder nstallationGuide pdf The installation guide can be found in the DeCyder installation via the start menu 28 9680 43 AA Addendum to DeCyder 2D User Manual 7 For local office contact information visit ce imagination at work and GE monogram are trademarks of General f Electric Company www gelifesciences com contact CyDye and DeCyder are trademarks of GE Healthcare companies DeCyder includes an Oracle 10g database Copyright 1995 2007 GE Healthcare Bio Sciences AB Oracle Corporation All rights reserved Bj rkgata n 30 All third party trademarks are the property of their respective owners 75184U ppsala Third Party Credit statements Swed en Oracle is a registered trademark of Oracle Corporation and or its affiliates This product includes the Xerces XML parser Copyright 2000 The Apache Software Foundation http www apache org All rights reserved www gelifesciences com d ecyder This product includes parts developed using ITK Copyright 1999 2003 Insight Software Consortium for licensing information see http www itk org Parts of
5. ill not be included when importing the workspace into the BVA module How to use spike normalization in DIA 1 Identify the spiked protein s using the gel view Select the spike proteins one by one and tick the Spike checkbox to indicate that this is a spiked protein C Pick CPIM Spike Pol Exclude Tip Use the comment field for the spiked protein name to have better control and be able to vary the assigned spiked proteins Addendum to DeCyder 2D 7 0 User Manual 28 9414 47 AA Details of new features in DIA module 2 Whenall spiked proteins are assigned click on the Process menu and select Normalize on Spiked Proteins to perform the spike normalization eee Help Process Gel Images Exclude Filter Normalize on Spiked Proteins Re Normalise Protein Filter 3 Verify the normalization by viewing the volume ratios for the spiked proteins one by one They should all have volume ratios close to 1 0 or 1 0 This is also shown in the histogram scatter plot but the values are close to 0 0 log 1 0 If a single spike protein is used the volume ratio Is 1 0 and the protein is placed on 0 0 in the histogram scatter plot The result of the normalization is displayed in the Histogram View with histograms showing the volume ratios If there is only one spike protein it will be in the middle of the graph at point 0 0 i e log 1 0 Compared to the model based normalization the pattern in the graph will re
6. main exactly the same but shifted to its spike normalized values Number of Spots Number of Spots b i h A oats ad ef 1 S ae Ti y i 35 2 4 0 8 0 6 1 9 3 2 45 ST 26 1415 0 0 13 26 38 51 64 Log Yolume Ratio Log Volume Ratio Figure 1 The histogram graph for a spike protein when normalized with model based normalization left and spike normalization right Note that the plot of volume ratios has the same pattern for both types of normalization but is shifted to the right for the spike normalization Note The menu item Process gt Re normalize is the same as before and will force a recalculation of the selected normalization Normally this is done automatically when changing the spike assignments but not when changing excluded spots 4 Addendum to DeCyder 2D User Manual 28 9680 43 AA Addendum to DeCyder 2D 7 0 User Manual 28 9414 47 AA Details of new features in BVA module Details of new features in BVA module In the BVA Main window there are two visible changes in version 7 1 e Inthe Data View Control Panel there is a new checkbox Spike e n the Protein table there is a new column Spike The word Spike in the new column indicates that the protein is assigned as a spike protein a0 9 jaa 711 AE a FII ct PALF Jir TIA sj FLLI l EE l At ik asl os TLIA EF pa 71319 jiz me 197 4s FLL 15 TILH F e A AEE g fe C Pte F Ph Dar Aii Pid ipi Fap
7. nt to select spike proteins with a minimum of interference overlay with any protein spots in the sample 2D map They should also be of other origin species than the sample proteins to avoid interference with the differential analysis or protein identification errors So called housekeeping proteins of the sample itself proteins known to be of constant concentration in all samples can also be used for the spike normalization procedure A typical application where the spike normalization is applicable is for the accurate determination of host cell proteins HCP in the downstream purification process for biopharmaceutical products Addendum to DeCyder 2D 7 0 User Manual 28 9414 47 AA Spike Normalization Detailed description of spike normalization Spike normalization is an alternative to the model based normalization It still normalizes volume ratios but based on a few spike proteins and not the complete set of proteins in a whole workspace The model based normalization is described in Appendix B 2 Detailed description of spot normalization procedure in DeCyder 2D 7 0 User Manual Spike normalization in DIA module In the DIA module spike normalization will adjust the volume ratio so the mean volume ratio for all spike proteins will be the 1 0 volume ratio The spike volume ratio is calculated as follows Ri V V Repike Where R is the spiked volume ratio for the i th protein V1 is the spot volume in one sample for the
8. o a separate license agreement with GE Healthcare 2010 General Electric Company All rights reserved First published Jan 2010 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them A copy of these terms and conditions is available on request Contact your local GE Healthcare representative for the most current information GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Japan Corporation Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan imagination at work 28 9680 43 AA 01 2010
9. standard corrected abundance for the spiked proteins one by one They should all have volume ratios close to 1 0 or 1 0 This is also shown in the graph the values should be close to 0 0 log 1 0 The line between the experimental groups is close to horizontal for all spiked proteins If a single spiked protein is used the volume ratio is 1 0 and the line is horizontal Model based normalization Spike normalization o 0 02 a ia oa 5 004 k 5 2 0 06 2 z z 9 0 08 cz D ja T ue 0 1 g T012 Hi i g _ l ja D E D 5 k 5 3 oO E D E Figure 2 The graph for a spike protein when normalized with model based normalization left and spike normalization right All spike proteins should have a graph between their experimental groups close to the 0 0 point i e log 1 0 Addendum to DeCyder 2D User Manual 28 9680 43 AA Addendum to DeCyder 2D 7 0 User Manual 28 9414 47 AA Installation of DeCyder 2D version 7 1 Installation of DeCyder 2D version 7 1 The installation guide can be found in the DeCyder installation via the start menu The procedure for upgrading is a bit different depending on whether DeCyder 2D 6 5 or 7 0 is already installed e f DeCyder 2D 6 5 is installed Backup the DeCyder database and then uninstall the complete software package see Section 5 Uninstallation in the DeCyderI nstallationGuide pdf before installing e f DeCyder 2D 7 0 is installed Backup th
10. this product implement the libTiff library Copyright 1988 1997 Sam Leffler Copyright 1991 1997 Silicon Graphics Inc http www libtiff org Parts of this product include NMath components from CenterSpace Software LLC Copyright 2002 2008 All rights reserved http www centerspace net Parts of this product include components from Farpoint Technologies Inc Copyright 1991 2007 All rights reserved http www fpoint com Parts of this product include the TeeChart component from Steema Software SL Copyright 1995 2008 All rights reserved http www teechart com Parts of this product include the open source MD5 implementation by L Deutsch Copyright 1999 2000 2002 Aladdin Enterprises All rights reserved Parts of this product include Cool Scrollbar Library Copyright 2001 J Brown CyDuye 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 CyDuye This product or portions thereof is manufactured under license from Carnegie Mellon University under US patent number US5 268 846 and other patents pending The purchase of CyDye fluors includes a 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 t
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