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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.
KeywordsHeart Isotopologue Monitoring Mouse MRM Multiple Multiplex Peptide Plasma Protein Proteomics Quantification Reaction
We are grateful to Genome Canada and Genome British Columbia for financial support (project codes 181MRM GAPP, 234DMP-DIG, 204PRO for operations and 214PRO for technology development). CHB is grateful for support from the Leading Edge Endowment Fund (University of Victoria) and for support from the Segal McGill Chair in Molecular Oncology at McGill University (Montreal, QC, Canada). CHB is also grateful for support from the Warren Y. Soper Charitable Trust and the Alvin Segal Family Foundation to the Jewish General Hospital (Montreal, QC, Canada).
We would like to thank Helena Petrosova for technical editing and Sarah A. Michaud for providing sample preparation protocols for mouse tissues. Helena Petrosova and Sarah A. Michaud are affiliated with the University of Victoria Genome BC Proteomics Centre.
- 7.LeBlanc A, Michaud SA, Percy AJ, Hardie DB, Yang J, Sinclair NJ, Proudfoot JI, Pistawka A, Smith DS, Borchers CH (2017) Multiplexed MRM-based protein quantitation using two different stable isotope-labeled peptide Isotopologues for calibration. J Proteome Res 16(7):2527–2536CrossRefPubMedGoogle Scholar
- 12.Percy AJ, Yang J, Chambers AG, Simon R, Hardie DB, Borchers CH (2014) Multiplexed MRM with internal standards for cerebrospinal fluid candidate protein biomarker quantitation. J Proteome Res 13(8):3733–3747Google Scholar
- 13.Kusebauch U, Campbell DS, Deutsch EW, Chu CS, Spicer DA, Brusniak MY, Slagel J, Sun Z, Stevens J, Grimes B, Shteynberg D, Hoopmann MR, Blattmann P, Ratushny AV, Rinner O, Picotti P, Carapito C, Huang CY, Kapousouz M, Lam H, Tran T, Demir E, Aitchison JD, Sander C, Hood L, Aebersold R, Moritz RL (2016) Human SRMAtlas: a resource of targeted assays to quantify the complete human proteome. Cell 166(3):766–778CrossRefPubMedPubMedCentralGoogle Scholar
- 16.Percy AJ, Michaud SA, Jardim A, Sinclair NJ, Zhang S, Mohammed Y, Palmer AL, Hardie DB, Yang J, LeBlanc AM, Borchers CH (2017) Multiplexed MRM-based assays for the quantitation of proteins in mouse plasma and heart tissue. Proteomics 17(7)Google Scholar