Antibody-based enrichment of peptides on magnetic beads for mass-spectrometry-based quantification of serum biomarkers
详细信息 查看全文
- 作者：Jeffrey R. Whiteaker ; Lei Zhao ; Heidi Y. Zhang ; Li-Chia Feng ; Brian D. Piening ; Leigh Anderson ; Am ; a G. Paulovich
- 关键词：Biomarkers ; Antipeptide antibody ; LC/MS/MS ; Targeted proteomics ; Stable isotope dilution ; Peptide quantitation
- 刊名：Analytical Biochemistry
- 出版年：2007
- 出版时间：1 March 2007
- 年：2007
- 卷：362
- 期：1
- 页码：44-54
- 全文大小：609 K
文摘
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 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.