Sequential multiplexed analyte quantification using peptide immunoaffinity enrichment coupled to mass spectrometry

Mol Cell Proteomics. 2012 Jun;11(6):M111.015347. doi: 10.1074/mcp.M111.015347. Epub 2011 Dec 27.

Abstract

Peptide immunoaffinity enrichment coupled to selected reaction monitoring (SRM) mass spectrometry (immuno-SRM) has emerged as a technology with great potential for quantitative proteomic assays. One advantage over traditional immunoassays is the tremendous potential for concurrent quantification of multiple analytes from a given sample (i.e. multiplex analysis). We sought to explore the capacity of the immuno-SRM technique for analyzing large numbers of analytes by evaluating the multiplex capabilities and demonstrating the sequential analysis of groups of peptides from a single sample. To evaluate multiplex analysis, immuno-SRM assays were arranged in groups of 10, 20, 30, 40, and 50 peptides using a common set of reagents. The multiplex immuno-SRM assays were used to measure synthetic peptides added to plasma covering several orders of magnitude concentration. Measurements made in large multiplex groups were highly correlated (r(2) ≥ 0.98) and featured good agreement (bias ≤ 1%) compared with single-plex assays or a 10-plex configuration. The ability to sequentially enrich sets of analyte peptides was demonstrated by enriching groups of 10 peptides from a plasma sample in a sequential fashion. The data show good agreement (bias ≤ 1.5%) and similar reproducibility regardless of enrichment order. These significant advancements demonstrate the utility of immuno-SRM for analyzing large numbers of analytes, such as in large biomarker verification experiments or in pathway-based targeted analysis.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Chromatography, Liquid
  • Humans
  • Immunosorbent Techniques
  • Mass Spectrometry
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification*
  • Peptide Mapping
  • Plasma / chemistry
  • Proteomics
  • Reproducibility of Results

Substances

  • Peptide Fragments