Offline Peptide Fractionation and Parallel Reaction Monitoring MS for the Quantitation of Low-Abundance Plasma Proteins

Methods Mol Biol. 2023:2628:353-364. doi: 10.1007/978-1-0716-2978-9_23.


Mass spectrometry (MS)-based protein quantitation is an attractive means for research and diagnostics due to its high specificity, precision, sensitivity, versatility, and the ability to develop multiplexed assays for the "absolute" quantitation of virtually any protein target. However, due to the large dynamic range of protein concentrations in blood, high abundance proteins in blood plasma hinder the detectability and quantification of lower-abundance proteins which are often relevant in the context of different diseases. Here we outline a streamlined method involving offline high-pH reversed-phase fractionation of human plasma samples followed by the quantitative analysis of specific fractions using nanoLC-parallel reaction monitoring (PRM) on a Q Exactive Plus mass spectrometer for peptide detection and quantitation with increased sensitivity. Because we use a set of synthetic peptide standards, we can more efficiently determine the precise retention times of the target peptides in the first-dimensional separation and specifically collect eluting fractions of interest for the subsequent targeted MS quantitation, making the analysis faster and easier. An eight-point standard curve was generated by serial dilution of a mixture of previously validated unlabeled ("light") synthetic peptides of interest at known concentrations. The corresponding heavy stable-isotope-labeled standard (SIS) analogues were used as normalizers to account for losses during sample processing and analysis. Using this method, we were able to improve the sensitivity of plasma protein quantitation by up to 50-fold compared to using nanoLC-PRM alone.

Keywords: 2D LC-PRM-MS; High-pH fractionation; Mass spectrometry; Parallel reaction monitoring; Plasma proteomics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blood Proteins / chemistry
  • Chemical Fractionation
  • Humans
  • Isotopes*
  • Mass Spectrometry / methods
  • Peptides* / chemistry


  • Peptides
  • Isotopes
  • Blood Proteins