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.
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 microg/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 microl 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.
Fortin
Tanguy
T
R&D Proteomique, bioMérieux SA, 69280 Marcy l'Etoile, France.
Salvador
Arnaud
A
Charrier
Jean Philippe
JP
Lenz
Cristof
C
Lacoux
Xavier
X
Morla
Aymeric
A
Choquet-Kastylevsky
Geneviève
G
Lemoine
Jérôme
J
D000595:Amino Acid Sequence; D015415:Biomarkers / Q000032:analysis / Q000737:chemistry; D002138:Calibration; D002853:Chromatography, Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D017430:Prostate-Specific Antigen / Q000032:analysis* / Q000097:blood / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012709:Serum Albumin; D053719:Tandem Mass Spectrometry
D015415:Biomarkers; D012709:Serum Albumin; D017430:Prostate-Specific Antigen