Multiplexed MRM-based Protein Quantitation Using Two Different Stable Isotope Labeled Peptides for Calibration
André LeBlanc, Sarah A. Michaud, Andrew J. Percy, Darryl B. Hardie, Juncong Yang, Nicholas J. Sinclair, Jillaine I. Proudfoot, Adam Pistawka, Derek S. Smith, and Christoph H. Borchers
Precise and robust quantitation of the endogenous plasma proteome is a requirement for fundamental and biomedical research as well as for clinical applications. 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 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) peptides 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.
Created on 4/13/17, 9:47 AM