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Originally published In Press as doi:10.1074/mcp.M500331-MCP200 on December 6, 2005.
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Molecular & Cellular Proteomics 5:573-588, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Quantitative Mass Spectrometric Multiple Reaction Monitoring Assays for Major Plasma Proteins*

Leigh Anderson{ddagger},§ and Christie L. Hunter

From the {ddagger} The Plasma Proteome Institute, Washington, D. C. 20009-3450 and Applied Biosystems, Foster City, California 94404

Quantitative LC-MS/MS assays were designed for tryptic peptides representing 53 high and medium abundance proteins in human plasma using a multiplexed multiple reaction monitoring (MRM) approach. Of these, 47 produced acceptable quantitative data, demonstrating within-run coefficients of variation (CVs) (n = 10) of 2–22% (78% of assays had CV <10%). A number of peptides gave CVs in the range 2–7% in five experiments (10 replicate runs each) continuously measuring 137 MRMs, demonstrating the precision achievable in complex digests. Depletion of six high abundance proteins by immunosubtraction significantly improved CVs compared with whole plasma, but analytes could be detected in both sample types. Replicate digest and depletion/digest runs yielded correlation coefficients (R2) of 0.995 and 0.989, respectively. Absolute analyte specificity for each peptide was demonstrated using MRM-triggered MS/MS scans. Reliable detection of L-selectin (measured at 0.67 µg/ml) indicates that proteins down to the µg/ml level can be quantitated in plasma with minimal sample preparation, yielding a dynamic range of 4.5 orders of magnitude in a single experiment. Peptide MRM measurements in plasma digests thus provide a rapid and specific assay platform for biomarker validation, one that can be extended to lower abundance proteins by enrichment of specific target peptides (stable isotope standards and capture by anti-peptide antibodies (SISCAPA)).

§ To whom correspondence should be addressed: The Plasma Proteome Inst., P. O. Box 53450, Washington, D. C. 20009-3450. Tel.: 301-728-1451; Fax: 202-234-9175; E-mail: leighanderson{at}plasmaproteome.org


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