Protein cleavage-isotope dilution mass spectrometry (PC-IDMS) can be used to quantify proteins, with an isotope-labeled analogue of the peptide fragment used as an internal standard. Here, we investigate use of a standard LC-MS/MS platform for quantifying a model biomarker directly from serum by this technique. We synthesized a peptide (IVGGWECEK) identical to the N-terminal tryptic fragment of PSA but with each glycine containing two 13C atoms and one 15N atom. PSA-free human serum was denatured with urea followed by the introduction of PSA standard and the stable isotope labeled internal standard peptide. The sample was then proteolyzed with trypsin and subjected to quantification using LC-MS/ MS on a triple quadrupole mass spectrometer. A linear least squares calibration curve made from five different concentrations of PSA added to serum and digested (each made in triplicate and randomly injected three times) had a mean slope of 0.973 (SE = 0.023), intercept of -0.003 (SE = 0.022), and R2 of 0.971. Recovery of calibrators ranged from 70 to 85% with a mean run-to-run CV of 13% and a mean within-run CV of 5.7%. PC-IDMS is a promising technique for quantifying proteins covering a broad range of applications from standardizing immunoassays to monitoring post-translational modifications to quantifying newly discovered biomarkers prior to the development and implementation of an immunoassay, just to name a few. Issues surrounding the application of PC-IDMS for the absolute quantification of proteins include selection of a proteolytic fragment for quantification that can be cleaved and isolated reproducibly over a broad dynamic range, stable isotope labeled synthetic peptide standards that give consistent results, and LC-MS/MS methods that provide adequate sensitivity and reproducibility without creating impractical analysis times. The results presented here show that absolute quantification can be performed on the model biomarker PSA introduced into denatured serum when analyzed by LC-MS/MS. However, concerns still exist regarding sensitivity compared to existing immunoassays as well as the reproducibility of PC-IDMS performed in different matrixes.
BACKGROUND Improved tests are needed for detection and management of prostate cancer. We hypothesized that differential gene expression in prostate tissue could help identify candidate blood biomarkers for prostate cancer and that blood from men with advanced prostate disease could be used to verify their presence in circulation. METHODS Candidate markers were identified using mRNA expression patterns from laser-capture microdissected prostate tissue. Tissue expression was confirmed using immunohistochemistry (IHC) for the subset of candidates having commercial antisera. Tissue extracts were analyzed with tandem mass spectrometry (MS/MS). Blood concentrations were measured using immunoassays and MS/MS of trypsin-digested, immuno-extracted peptides. RESULTS Thirty-five novel candidate prostate adenocarcinoma biomarkers were selected. Tissue expression was confirmed for all of the 13 markers having commercial antisera for IHC and six of these markers showed statistical discrimination between normal and malignant tissue. Only 5 of these markers were detected in tissue extracts using MS/MS. Sixteen of the 35 candidate markers were successfully assayed in blood. Four of eight biomarkers measured with ELISA and 3 of 10 biomarkers measured by targeted MS showed statistically significant increases in blood concentrations of advanced prostate cancer cases, compared to controls. CONCLUSION Seven novel biomarkers identified by gene expression profiles in prostate tissue were shown to have statistically significant increased levels in blood from men with advanced prostate adenocarcinoma compared to controls: APOC1, ASPN, COMP, CXCL11, CXCL9, F5, and PCSK6.
Background: Human glandular kallikrein (hK2) is a serine protease that has 79% amino acid identity with prostate-specific antigen (PSA). Both free hK2 and hK2 complexed to α1-antichymotrypsin (ACT) are present in the blood in low concentrations. We wished to measure hK2 in serum with limited contribution from hK2-ACT for the results. Methods: We developed an automated assay for hK2 with use of a select pair of monoclonal antibodies. The prototype assay was implemented on a Beckman Coulter ACCESS® analyzer. Results: The detection limit of the assay was 1.5 ng/L, the “functional sensitivity” (day-to-day CV <15%) was <4 ng/L, cross-reactivity with PSA and PSA-ACT was negligible, and cross-reactivity with hK2-ACT was 2%. After surgical removal of prostate glands, serum hK2 was <7 ng/L and was <15 ng/L in most healthy women. The median serum concentration of hK2 in healthy men without prostate cancer was 26 ng/L. The median concentration of hK2 was 72 ng/L for men having prostate cancer with lower Gleason scores compared with 116 ng/L for men with more advanced cancer. The concentration of hK2 correlated weakly with PSA, with the mean hK2 concentrations generally 30- to 80-fold lower than PSA concentrations. Conclusion: The availability of a robust, high sensitivity automated assay for hK2 should facilitate further investigations of the role of hK2 measurements in the management of patients with prostate disease.
Background: Human kallikrein 2 (hK2) shares 80% sequence identity with prostate-specific antigen (PSA). Because both hK2 and hK2-α1-antichymotrypsin (hK2-ACT) complexes have been identified in patient sera, we devised an immunoassay for total hK2 [(thK2); hK2 and hK2-ACT] and evaluated it in healthy subjects and patients with prostate disease. Methods: We developed monoclonal antibodies (mAbs) with high specificity for hK2 and hK2-ACT and minimal cross-reactivity to PSA. Using these mAbs, a sandwich assay was developed and its specificity for forms of hK2 was assessed. Serum samples (n = 1035) from healthy volunteers, patients with increased PSA, and men who had undergone radical prostatectomy were assayed for thK2. We also measured thK2 in samples before and after storage under common laboratory conditions. Results: The minimum detectable concentration in the thK2 assay was 0.008 μg/L, and PSA cross-reactivity was <0.001%. The assay detected prohK2 and three different hK2–serum protease complexes. The median serum concentration of thK2 in control samples (0.013 μg/L) was significantly lower than the median in samples from patients with increased PSA concentrations (0.085 μg/L). Immunoreactive hK2 changed little in samples stored for up to 1 month at −70 °C. Conclusions: The thK2 assay recognizes all forms of hK2 that have been found in bodily fluids to date.
Mass spectrometry quantitation of peptides derived from trypsin digestion of immune-extracted PSA could be used to harmonize PSA immunoassays.
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