Multiple Reaction Monitoring Using Double Isotopologue Peptide Standards for Protein Quantification

Multiple reaction monitoring (MRM) is a technique used in tandem mass spectrometry where the first mass analyzer preselects parent ions for fragmentation and the second mass analyzer transmits selected product ions to the detector. This targeted technique has found widespread application in bottom-up proteomics for monitoring target peptides in a complex enzymatic digest. Quantitative MRM can be performed on enzymatically digested samples using spiked-in synthetic peptide standards, providing unsurpassed quantitative accuracy and a dynamic range of four orders of magnitude, often eliminating the need for prior depletion of high-abundance proteins. The development of MRM assays requires technical rigor, and this chapter details a methodology for sample preparation, data acquisition, and analyses to successfully perform quantitative MRM assays using two distinct isotopologue peptide standards to quantify proteins in mouse plasma and heart tissue.