Enhanced sensitivity for selected reaction monitoring mass spectrometry-based targeted proteomics using a dual stage electrodynamic ion funnel interface

Mol Cell Proteomics. 2011 Feb;10(2):M000062-MCP201. doi: 10.1074/mcp.M000062-MCP201. Epub 2010 Apr 21.

Abstract

Selected reaction monitoring mass spectrometry (SRM-MS) is playing an increasing role in quantitative proteomics and biomarker discovery studies as a method for high throughput candidate quantification and verification. Although SRM-MS offers advantages in sensitivity and quantification compared with other MS-based techniques, current SRM technologies are still challenged by detection and quantification of low abundance proteins (e.g. present at ∼10 ng/ml or lower levels in blood plasma). Here we report enhanced detection sensitivity and reproducibility for SRM-based targeted proteomics by coupling a nanospray ionization multicapillary inlet/dual electrodynamic ion funnel interface to a commercial triple quadrupole mass spectrometer. Because of the increased efficiency in ion transmission, significant enhancements in overall signal intensities and improved limits of detection were observed with the new interface compared with the original interface for SRM measurements of tryptic peptides from proteins spiked into non-depleted mouse plasma over a range of concentrations. Overall, average SRM peak intensities were increased by ∼70-fold. The average level of detection for peptides also improved by ∼10-fold with notably improved reproducibility of peptide measurements as indicated by the reduced coefficients of variance. The ability to detect proteins ranging from 40 to 80 ng/ml within mouse plasma was demonstrated for all spiked proteins without the application of front-end immunoaffinity depletion and fractionation. This significant improvement in detection sensitivity for low abundance proteins in complex matrices is expected to enhance a broad range of SRM-MS applications including targeted protein and metabolite validation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Biomarkers / metabolism
  • Cattle
  • Chickens
  • Cytochromes c / metabolism
  • Escherichia coli / enzymology
  • Horses / metabolism
  • Mass Spectrometry / methods*
  • Mice
  • Muscle, Skeletal / metabolism
  • Myoglobin / metabolism
  • Ovalbumin / metabolism
  • Peptides / chemistry
  • Proteomics / methods*
  • Trypsin / chemistry

Substances

  • Biomarkers
  • Myoglobin
  • Peptides
  • Ovalbumin
  • Cytochromes c
  • Trypsin