Standard Operating Procedures and Protocols for the Preparation and Analysis of Plasma Samples Using the iTRAQ Methodology
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The Applied Biosystems iTRAQ technique is based on derivatization of peptide samples with mass-balanced tags. The samples are then combined, and quantitation is based on reporter ion abundances in the MS/MS spectrum. The iTRAQ technique, with its capability for the analysis of either 4 or 8 samples in a single LC-MS/MS analysis, is one of the most powerful techniques available today for “shotgun” quantitative proteomics. However, it is not an easy technique, and involves multiple – and critical – digestion, separation, and derivatization steps. Our laboratory has spent several years developing over 40 standard operating procedures (SOPs) for the generation of reproducible iTRAQ data. These protocols are designed for generating and analyzing iTRAQ data on the Applied Biosystems 4800 MALDI-TOF/TOF instrument and the Applied Biosystems QSTAR ESI-MS/MS, which are two of the most commonly-used instruments for iTRAQ analysis. We have also included sections on the Applied Biosystems ProteinPilot software which is used for analyzing the data and obtaining protein expression ratios. Standard operating procedures are obviously designed to be used with these specific instruments. For laboratories with other instruments, we have tried to include sufficient annotation and descriptions of the procedures so that these protocols can be adapted for use on other equipment. Likewise, many of these protocols are not specific to iTRAQ, and can be used for sample preparation and analysis for other assays as well. For more complete and detailed protocols, the reader is referred to the on-line supplementary information on our website at http://www.proteincentre.com/itraq-book-chapter-supplemental-information.
KeywordsiTRAQ LC-ESI-MS/MS LC-MALDI Protein quantification SCX
The authors would like to thank the organizations for funding work relevant to these methods including the PROOF Centre of Excellence, Genome Canada, Novartis, IBM, Genome BC, University of British Columbia VP Research, St. Paul’s Hospital Foundation, BC Transplant, and the James Hogg iCAPTURE Centre. We would also like to thank Drs. Steven Parnell and Oscar Alzate for critical reading of this manuscript.
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