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Anal Biochem. 2007 Mar 1;362(1):44-54. Epub 2006 Dec 20.

Antibody-based enrichment of peptides on magnetic beads for mass-spectrometry-based quantification of serum biomarkers.

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1
Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N., PO Box 19024, Seattle, WA 98109-1024, USA.

Abstract

A major bottleneck for validation of new clinical diagnostics is the development of highly sensitive and specific assays for quantifying proteins. We previously described a method, stable isotope standards with capture by antipeptide antibodies, wherein a specific tryptic peptide is selected as a stoichiometric representative of the protein from which it is cleaved, is enriched from biological samples using immobilized antibodies, and is quantitated using mass spectrometry against a spiked internal standard to yield a measure of protein concentration. In this study, we optimized a magnetic-bead-based platform amenable to high-throughput peptide capture and demonstrated that antibody capture followed by mass spectrometry can achieve ion signal enhancements on the order of 10(3), with precision (CVs <10%) and accuracy (relative error approximately 20%) sufficient for quantifying biomarkers in the physiologically relevant ng/mL range. These methods are generally applicable to any protein or biological fluid of interest and hold great potential for providing a desperately needed bridging technology between biomarker discovery and clinical application.

PMID:
17241609
PMCID:
PMC1852426
DOI:
10.1016/j.ab.2006.12.023
[Indexed for MEDLINE]
Free PMC Article

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