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

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

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  • 1Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA.

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.

PMID:
22949669
[PubMed - indexed for MEDLINE]
PMCID:
PMC3458402
Free PMC Article
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