Multiplexed MRM-Based Protein Quantitation Using Two Different Stable Isotope-Labeled Peptide Isotopologues for Calibration

• Published in: Journal of proteome research Vol. 16; no. 7; pp. 2527 - 2536
• Main Authors: LeBlanc, André, Michaud, Sarah A, Percy, Andrew J, Hardie, Darryl B, Yang, Juncong, Sinclair, Nicholas J, Proudfoot, Jillaine I, Pistawka, Adam, Smith, Derek S, Borchers, Christoph H
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 07.07.2017
• Subjects: Amino Acid Sequence; Amino Acids - chemistry; Biological Assay; Blood Proteins - chemistry; Blood Proteins - standards; Calibration; Carbon Isotopes; Chromatography, Liquid - standards; Humans; Isotope Labeling - methods; Mass Spectrometry - standards; Nitrogen Isotopes; Peptides - blood; Peptides - standards; Proteomics - methods; Proteomics - standards; Reference Standards; Staining and Labeling - methods; peptide assay validation; peptide quantitation; method development; MRM; surrogate standard; peptide calibration; stable isotope-labeled peptides; peptide isotopologues; surrogate matrix
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/acs.jproteome.7b00094

When quantifying endogenous plasma proteins for fundamental and biomedical research − as well as for clinical applications − precise, reproducible, and robust assays are required. Targeted detection of peptides in a bottom-up strategy is the most common and precise mass spectrometry-based quantitation approach when combined with the use of stable isotope-labeled peptides. However, when measuring protein in plasma, the unknown endogenous levels prevent the implementation of the best calibration strategies, since no blank matrix is available. Consequently, several alternative calibration strategies are employed by different laboratories. In this study, these methods were compared to a new approach using two different stable isotope-labeled standard (SIS) peptide isotopologues for each endogenous peptide to be quantified, enabling an external calibration curve as well as the quality control samples to be prepared in pooled human plasma without interference from endogenous peptides. This strategy improves the analytical performance of the assay and enables the accuracy of the assay to be monitored, which can also facilitate method development and validation.