Antibody-free, targeted mass-spectrometric approach for quantification of proteins at low picogram per milliliter levels in human plasma/serum

Proc Natl Acad Sci U S A. 2012 Sep 18;109(38):15395-400. doi: 10.1073/pnas.1204366109. Epub 2012 Sep 4.

Abstract

Sensitive detection of low-abundance proteins in complex biological samples has typically been achieved by immunoassays that use antibodies specific to target proteins; however, de novo development of antibodies is associated with high costs, long development lead times, and high failure rates. To address these challenges, we developed an antibody-free strategy that involves PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing) for sensitive selected reaction monitoring (SRM)-based targeted protein quantification. The strategy capitalizes on high-resolution reversed-phase liquid chromatographic separations for analyte enrichment, intelligent selection of target fractions via on-line SRM monitoring of internal standards, and fraction multiplexing before nano-liquid chromatography-SRM quantification. Application of this strategy to human plasma/serum demonstrated accurate and reproducible quantification of proteins at concentrations in the 50-100 pg/mL range, which represents a major advance in the sensitivity of targeted protein quantification without the need for specific-affinity reagents. Application to a set of clinical serum samples illustrated an excellent correlation between the results obtained from the PRISM-SRM assay and those from clinical immunoassay for the prostate-specific antigen level.

Publication types

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

MeSH terms

  • Animals
  • Antibodies / chemistry*
  • Biomarkers / metabolism
  • Blood Proteins / analysis
  • Calibration
  • Carbonic Anhydrases / blood
  • Cattle
  • Chromatography, Liquid / methods
  • Female
  • Humans
  • Mass Spectrometry / methods*
  • Prostate-Specific Antigen / blood
  • Proteins / analysis*
  • Proteomics / methods
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Systems Biology / methods

Substances

  • Antibodies
  • Biomarkers
  • Blood Proteins
  • Proteins
  • Prostate-Specific Antigen
  • Carbonic Anhydrases