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.

Mass spectrometry-based strategies for the quantification of low-abundance putative protein biomarkers in human blood currently require extensive sample fractionation steps which hamper their implementation in a routine and robust way across clinical laboratories. We demonstrate that a technique using MS(3) reconstructed chromatograms on a signature of secondary ions issued from a trapped primary product ion, termed multiple reaction monitoring cubed (MRM(3)), enables targeting protein biomarkers in the low nanogram/milliliter range in nondepleted human serum. The simple two-step workflow is based on a trypsin proteolysis of whole serum (100 microL) followed by enrichment of targeted proteotypic peptides on a solid phase extraction column using mixed-cation exchange resin. MRM(3)'s fidelity of peak detection extends the dynamic range and limit of quantitation (LOQ) of protein biomarkers to the low nanogram/milliliter range, corresponding to a concentration that is 10(6)-fold lower than the concentration of the most abundant proteins in serum. The power of the MRM(3) method is illustrated by the assay of prostate specific antigen in nondepleted human sera of patients. The results correlate well with the established method for determining PSA levels in serum, i.e., enzyme-linked immunosorbent assay (ELISA) tests.

Mass spectrometry (MS) in Selected Reaction Monitoring (SRM) mode is proposed for in-depth characterisation of microorganisms in a multiplexed analysis. Within 60–80 minutes, the SRM method performs microbial identification (I), antibiotic-resistance detection (R), virulence assessment (V) and it provides epidemiological typing information (T). This SRM application is illustrated by the analysis of the human pathogen Staphylococcus aureus, demonstrating its promise for rapid characterisation of bacteria from positive blood cultures of sepsis patients.