title: Serum concentrations of prostate-specific antigen measured using immune extraction, trypsin digestion, and tandem mass spectrometry quantification of LSEPAELTDAVK peptide.
abstract: BACKGROUND
  Prostate-specific antigen (PSA) is a 34-kDa glycoprotein with chymotrypsin-like enzyme activity that circulates both in free forms and complexed to various enzyme inhibitors including antichymotrypsin and α2-macroglobulin. Prostate-specific antigen bound to α2-macroglobulin is not detected by commercial PSA immunoassays.
  
  
  OBJECTIVE
  To develop a mass spectrometry assay that detects the same forms of PSA as the immunoassays, which could serve as a reference for harmonizing PSA immunoassays.
  
  
  METHODS
  Prostate-specific antigen was immune extracted from serum, trypsin was digested, and the LSEPAELTDAVK peptide was quantitated on an API 5000 spectrometer. Calibrators were made by adding 10% free and 90% antichymotrypsin-bound PSA to female sera. The assay was standardized to the World Health Organization 96/670 reference standard. Validation of clinical utility and comparisons with 2 immunoassays (Roche cobas and Beckman Access) were performed using frozen sera aliquots from 100 men undergoing prostate biopsy (50 negative, 50 with cancer) and 5 serial samples collected over time from 5 men with advanced prostate cancer.
  
  
  RESULTS
  The antibody extraction efficiency was greater than 99%. The assay has an analytic range from 1.2 to 76 ng/mL, with precision ranging from 8.6% at 1.5 ng/mL to 5.4% at 27 ng/mL. The mass spectrometry assay correlated well with 2 immunoassays. All 3 assays showed statistically equivalent separation of prostate cancer from benign disease using receiver operating characteristic curve analysis.
  
  
  CONCLUSIONS
  This mass spectrometry assay can reliably measure PSA concentrations in human serum and could serve as a reference standard for harmonizing PSA immunoassays.
authors.affiliation: From the Departments of Health Sciences Research (Dr E. Klee and Mr Bergstralh) and Laboratory Medicine and Pathology (Drs Bondar, Trushin, Singh, and G. Klee and Ms. Goodmanson), Mayo Clinic College of Medicine, Rochester, Minnesota; and the Plasma Proteome Institute, Washington, DC (Dr Anderson).
authors.forename: Eric W
authors.initials: EW
authors.lastname: Klee
authors.forename: Olga P
authors.initials: OP
authors.lastname: Bondar
authors.forename: Marcia K
authors.initials: MK
authors.lastname: Goodmanson
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Trushin
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Bergstralh
authors.forename: Ravinder J
authors.initials: RJ
authors.lastname: Singh
authors.forename: N Leigh
authors.initials: NL
authors.lastname: Anderson
authors.forename: George G
authors.initials: GG
authors.lastname: Klee
mesh_terms: D053000:Analytic Sample Preparation Methods; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002138:Calibration; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry; D008297:Male; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017430:Prostate-Specific Antigen / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000097:blood* / Q000175:diagnosis; D059748:Proteolysis; D012372:ROC Curve; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D007202:Indicators and Reagents; D010446:Peptide Fragments; D014357:Trypsin; D017430:Prostate-Specific Antigen