Journal
ANALYTICAL BIOCHEMISTRY
Volume 362, Issue 1, Pages 44-54Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2006.12.023
Keywords
biomarkers; antipeptide antibody; LC/MS/MS; targeted proteomics; stable isotope dilution; peptide quantitation
Funding
- PHS HHS [23XS144A, 23 XS144A] Funding Source: Medline
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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 103, with precision (CVs < 10%) and accuracy (relative error similar to 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. (c) 2006 Elsevier Inc. All rights reserved.
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