NDLI: An Automated and Multiplexed Method for High Throughput Peptide Immunoaffinity Enrichment and Multiple Reaction Monitoring Mass Spectrometry-based Quantification of Protein Biomarkers*

Content Provider	PubMed Central
Author	Whiteaker, Jeffrey R. Zhao, Lei Anderson, Leigh Paulovich, Amanda G.
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. Stable isotope standards with capture by anti-peptide antibodies (SISCAPA) has shown tremendous potential to meet this need by combining peptide immunoaffinity enrichment with quantitative mass spectrometry. In this study, 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 μl of plasma) with sufficient precision (median coefficient of variation, 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.
Related Links	http://dx.doi.org/10.1074/mcp.m900254-mcp200
Ending Page	196
Page Count	13
Starting Page	184
File Format	PDF
ISSN	15359476
e-ISSN	15359484
Journal	Molecular & Cellular Proteomics : MCP
Issue Number	1
Volume Number	9
Language	English
Publisher	The American Society for Biochemistry and Molecular Biology
Publisher Date	2010-01-01
Access Restriction	Open
Rights Holder	The American Society for Biochemistry and Molecular Biology
Subject Keyword	Analytical Chemistry Biochemistry Molecular Biology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Medicine Analytical Chemistry Molecular Biology Biochemistry

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