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Molecular & Cellular Proteomics 8:1006-1015, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Clinical Quantitation of Prostate-specific Antigen Biomarker in the Low Nanogram/Milliliter Range by Conventional Bore Liquid Chromatography-Tandem Mass Spectrometry (Multiple Reaction Monitoring) Coupling and Correlation with ELISA Tests

Tanguy Fortin^,, Arnaud Salvador, Jean Philippe Charrier, Cristof Lenz^¶, Xavier Lacoux, Aymeric Morla, Geneviève Choquet-Kastylevsky and Jérôme Lemoine^,||

From the R&D Proteomique, bioMérieux SA, 69280 Marcy l’Etoile, France, UMR 5180 Sciences Analytiques, Université de Lyon, Lyon 1, 69622 Villeurbanne Cedex, France, and ^¶ Proteomic and Small Molecules Support, Applied Biosystems, 64293 Darmstadt, Germany

Proteomics discovery leads to a list of potential protein biomarkers that have to be subsequently verified and validated with a statistically viable number of patients. Although the most sensitive, the development of an ELISA test is time-consuming when antibodies are not available and need to be conceived. Mass spectrometry analysis driven in quantitative multiple reaction monitoring mode is now appearing as a promising alternative to quantify proteins in biological fluids. However, all the studies published to date describe limits of quantitation in the low µg/ml range when no immunoenrichment of the target protein is applied, whereas the concentration of known clinical biomarkers is usually in the ng/ml range. Using prostate-specific antigen as a model biomarker, we now provide proof of principle that mass spectrometry enables protein quantitation in a concentration range of clinical interest without immunoenrichment. We have developed and optimized a robust sample processing method combining albumin depletion, trypsin digestion, and solid phase extraction of the proteotypic peptides starting from only 100 µl of serum. For analysis, mass spectrometry was coupled to a conventional liquid chromatography system using a 2-mm-internal diameter reverse phase column. This mass spectrometry-based strategy was applied to the quantitation of prostate-specific antigen in sera of patients with either benign prostate hyperplasia or prostate cancer. The quantitation was performed against an external calibration curve by interpolation, and results showed good correlation with existing ELISA tests applied to the same samples. This strategy might now be implemented in any clinical laboratory or certified company for further evaluation of any putative biomarker in the low ng/ml range of serum or plasma.

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