An automated and multiplexed method for high throughput peptide immunoaffinity enrichment and multiple reaction monitoring mass spectrometry-based quantification of protein biomarkers Export

@article{citeulike:6086576, abstract = {There is an urgent need for quantitative assays in verifying and validating the large numbers of protein biomarker candidates produced in modern "-omics" experiments. SISCAPA (Stable Isotope Standards with Capture by Anti-Peptide Antibodies) has shown tremendous potential to meet this need by combining peptide immunoaffinity enrichment with quantitative mass spectrometry. In this paper, we describe three significant advances to the SISCAPA technique. First, we develop a method for an automated magnetic bead-based platform capable of high throughput processing. Second, we implement the automated method in a multiplexed SISCAPA assay (nine targets in one assay) and assess the performance characteristics of the multiplexed assay. Using the automated, multiplexed platform we demonstrate detection limits in the physiologically relevant ng/mL range (from 10 [mu]L plasma), with sufficient precision (median CV 12.6\%) for quantifying biomarkers. Third, we demonstrate that enrichment of peptides from larger volumes of plasma (1 mL) can extend the limits of detection to the low pg/mL range of protein concentration. The method is generally applicable to any protein or biological specimen of interest and holds great promise for analyzing large numbers of biomarker candidates. 10.1074/mcp.M900254-MCP200}, author = {Whiteaker, Jeffrey R. and Zhao, Lei and Anderson, Leigh and Paulovich, Amanda G.}, citeulike-article-id = {6086576}, citeulike-linkout-0 = {http://dx.doi.org/10.1074/mcp.M900254-MCP200}, citeulike-linkout-1 = {http://www.mcponline.org/content/M900254-MCP200v1/.abstract}, citeulike-linkout-2 = {http://www.mcponline.org/content/M900254-MCP200v1/.full.pdf}, citeulike-linkout-3 = {http://view.ncbi.nlm.nih.gov/pubmed/19843560}, citeulike-linkout-4 = {http://www.hubmed.org/display.cgi?uids=19843560}, day = {20}, doi = {10.1074/mcp.M900254-MCP200}, journal = {Mol Cell Proteomics}, keywords = {antibody, mrm, plasma, proteomics}, month = {October}, pages = {M900254-MCP200+}, posted-at = {2009-11-08 19:55:38}, priority = {2}, title = {An automated and multiplexed method for high throughput peptide immunoaffinity enrichment and multiple reaction monitoring mass spectrometry-based quantification of protein biomarkers}, url = {http://dx.doi.org/10.1074/mcp.M900254-MCP200}, year = {2009} }

TY - JOUR ID - citeulike:6086576 L3 - citeulike-article-id:6086576 N2 - There is an urgent need for quantitative assays in verifying and validating the large numbers of protein biomarker candidates produced in modern "-omics" experiments. SISCAPA (Stable Isotope Standards with Capture by Anti-Peptide Antibodies) has shown tremendous potential to meet this need by combining peptide immunoaffinity enrichment with quantitative mass spectrometry. In this paper, we describe three significant advances to the SISCAPA technique. First, we develop a method for an automated magnetic bead-based platform capable of high throughput processing. Second, we implement the automated method in a multiplexed SISCAPA assay (nine targets in one assay) and assess the performance characteristics of the multiplexed assay. Using the automated, multiplexed platform we demonstrate detection limits in the physiologically relevant ng/mL range (from 10 [mu]L plasma), with sufficient precision (median CV 12.6%) for quantifying biomarkers. Third, we demonstrate that enrichment of peptides from larger volumes of plasma (1 mL) can extend the limits of detection to the low pg/mL range of protein concentration. The method is generally applicable to any protein or biological specimen of interest and holds great promise for analyzing large numbers of biomarker candidates. 10.1074/mcp.M900254-MCP200 JF - Mol Cell Proteomics TI - An automated and multiplexed method for high throughput peptide immunoaffinity enrichment and multiple reaction monitoring mass spectrometry-based quantification of protein biomarkers SP - M900254-MCP200 KW - antibody KW - mrm KW - plasma KW - proteomics AU - Whiteaker, Jeffrey AU - Zhao, Lei AU - Anderson, Leigh AU - Paulovich, Amanda PY - 2009/10/20/ UR - http://dx.doi.org/10.1074/mcp.M900254-MCP200 ER -