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

Abstract

Geneviè ve Choquet-Kastylevsky ‡, and Jé rô me Lemoine §ʈProteomics 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 g/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 l 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.

The Bottleneck in the Cancer Biomarker Pipeline and Protein Quantification through Mass Spectrometry–Based Approaches: Current Strategies for Candidate Verification

“…proteins in both single (33)(34)(35)(36)(37)(38)(39)(40) and multiplexed assay formats (41)(42)(43)(44). The proteins quantified in these studies are summarized in Table 1.…”

“…In general, direct plasma or serum digests without prior attempts to minimize sample complexity have resulted in low milligram per liter quantification ranges at best (33,43,45 ). For instance, a number of different studies that have quantified prostate-specific antigen (PSA) through coupling of various sample purification strategies have generally shown that quantification improves from the low milligram per liter range in untreated plasma analysis (33 ) to the low microgram per liter range with prior depletion and fractionation (39,41 ) and the low to sub-microgram per liter range with prior immunoextration (46 ) ( Table 2). Although various confounding factors such as variations in instrumentation used in the different laboratories undoubtedly contributed to some of the differences observed, this trend can also be seen within the same study (Table 2) (41,46 ).…”

Applications of targeted proteomics in systems biology and translational medicine

“…Keshishian and coworkers developed multiplexed assays for six plasma proteins present in 1–10 ng/ml using strong cation exchange chromatography and major abundant protein depletion, but without immunoaffininty enrichment, demonstrating that the abundance range of typical candidate biomarkers (ng/ml) is achievable with targeted proteomics 82. Fortin and coworkers also achieved ng/ml sensitivity without immunoaffinity enrichment by quantitating prostate‐specific antigen (PSA) in sera from patients with prostate cancer or benign hyperplasia; moreover, their MS results agreed with established ELISA tests for PSA 83. Shi and coworkers used an antibody‐free approach termed PRISM (high‐pressure, high‐resolution separations coupled with intelligent selection and multiplexing) in order to quantitate PSA levels in the range of 50–100 pg/ml, also with excellent correlation to clinical immunoassays 84.…”

Mass spectrometry: a revolution in clinical microbiology?

“…The LC-ESI-QqQ in selected or multiple reactions way has been used for routine detection of small molecules including metabolites and drugs [31] . More recently, the LC-ESI-QqQ has been suggested as a replacement for classical ELISAs for the quantitation of proteins in complex matrices [98] . The MALDI triple quadruple measures enzyme-mediated, time-dependent hydrolysis of the β -lactam ring structure of penicillin G and ampicillin and inhibition of hydrolysis by clavulanic acid for clavulanic acid susceptible β -lactamases.…”