Antibody-based Enrichment of Peptides on Magnetic Beads for Mass-spectrometry-based Quantification of Serum Biomarkers

Overview

Journal Anal Biochem

Publisher Elsevier

Specialty Biochemistry

Date 2007 Jan 24

PMID 17241609

Citations 87

Authors

Jeffrey R Whiteaker

Lei Zhao

Heidi Y Zhang

Li-Chia Feng

Brian D Piening

Leigh Anderson

Amanda G Paulovich

Affiliations

Soon will be listed here.

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.

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