Basics and Practices of Quantitative Proteomics Using Selected Reaction Monitoring (SRM)

Abstract

Selected reaction monitoring mode (SRM)-based strategies have recently been developed for quantitation of proteins. Using sequence information from the proteins, these strategies can be used to develop simultaneous quantitative methods for several proteins of interest. In this article, we discussed the problems and potential roles of SRM-based strategies in protein quantitation. “Absolute” quantitation of proteins is one of the strengths of SRM strategies. For mass spectrometry, protein samples should be digested with enzymes to their component peptides. But we cannot correct for the sample loss or efficiency of enzyme digestion in this preparatory step. Therefore, quantitative values from SRM strategies may underestimate protein abundance. In this article, we introduced our solution to this problem with isotope-labeled protein as quantitative reference. Also, we have developed a PTM profiling method with isotope-labeled full-length proteins as references. This method makes it possible to obtain the modification sites and rates of proteins using differential analysis for the quantitative values of each peptide.