Serum concentrations of prostate-specific antigen measured using immune extraction, trypsin digestion, and tandem mass spectrometry quantification of LSEPAELTDAVK peptide

Eric W Klee, Olga P. Bondar, Marcia K. Goodmanson, Sergey A. Trushin, Eric J. Bergstralh, Ravinder Jit Singh, N. Leigh Anderson, George G. Klee

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Context. - 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. Design. - 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.

Original languageEnglish (US)
Pages (from-to)1381-1386
Number of pages6
JournalArchives of Pathology and Laboratory Medicine
Volume138
Issue number10
DOIs
StatePublished - Oct 1 2014

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Prostate-Specific Antigen
Tandem Mass Spectrometry
Trypsin
Digestion
Immunoassay
Peptides
Serum
Mass Spectrometry
Macroglobulins
Prostatic Neoplasms
Enzyme Inhibitors
ROC Curve
Immune Sera
Prostate
Glycoproteins
Biopsy
Antibodies

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Medical Laboratory Technology
  • Medicine(all)

Cite this

Serum concentrations of prostate-specific antigen measured using immune extraction, trypsin digestion, and tandem mass spectrometry quantification of LSEPAELTDAVK peptide. / Klee, Eric W; Bondar, Olga P.; Goodmanson, Marcia K.; Trushin, Sergey A.; Bergstralh, Eric J.; Singh, Ravinder Jit; Anderson, N. Leigh; Klee, George G.

In: Archives of Pathology and Laboratory Medicine, Vol. 138, No. 10, 01.10.2014, p. 1381-1386.

Research output: Contribution to journalArticle

Klee, Eric W ; Bondar, Olga P. ; Goodmanson, Marcia K. ; Trushin, Sergey A. ; Bergstralh, Eric J. ; Singh, Ravinder Jit ; Anderson, N. Leigh ; Klee, George G. / Serum concentrations of prostate-specific antigen measured using immune extraction, trypsin digestion, and tandem mass spectrometry quantification of LSEPAELTDAVK peptide. In: Archives of Pathology and Laboratory Medicine. 2014 ; Vol. 138, No. 10. pp. 1381-1386.
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abstract = "Context. - 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. Design. - 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.",
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AU - Klee, Eric W

AU - Bondar, Olga P.

AU - Goodmanson, Marcia K.

AU - Trushin, Sergey A.

AU - Bergstralh, Eric J.

AU - Singh, Ravinder Jit

AU - Anderson, N. Leigh

AU - Klee, George G.

PY - 2014/10/1

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N2 - Context. - 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. Design. - 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.

AB - Context. - 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. Design. - 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.

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