U.S. Department of Energy

Pacific Northwest National Laboratory

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

TitleAntibody-free, targeted mass-spectrometric approach for quantification of proteins at low picogram per milliliter levels in human plasma/serum.
Publication TypeJournal Article
Year of Publication2012
AuthorsShi T, Fillmore TL, Sun X, Zhao R, Schepmoes AA, Hossain M, Xie F, Wu S, Kim J-S, Jones N, Moore RJ, Pasa-Tolic L, Kagan J, Rodland KD, Liu T, Tang K, Camp DG, Smith RD, Qian W-J
JournalProc Natl Acad Sci U S A
KeywordsAnimals, Antibodies, Biological Markers, Blood Proteins, Calibration, Carbonic Anhydrases, Cattle, Chromatography, Liquid, Female, Humans, Mass Spectrometry, Prostate-Specific Antigen, Proteins, Proteomics, Reproducibility of Results, Sensitivity and Specificity, Systems Biology
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.

DOI10.1073/pnas.1204366109
PubMed ID22949669
PubMed Central IDPMC3458402
Grant List5P41 RR018522 / RR / NCRR NIH HHS / United States
8P41 GM103493 / GM / NIGMS NIH HHS / United States
CA111244 / CA / NCI NIH HHS / United States
DP2OD006668 / OD / NIH HHS / United States
P41 GM103493 / GM / NIGMS NIH HHS / United States
P41 RR018522 / RR / NCRR NIH HHS / United States
U24 CA160019 / CA / NCI NIH HHS / United States
U24-CA-160019 / CA / NCI NIH HHS / United States
Y01-CN-05013-29 / CN / NCI NIH HHS / United States
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