Selected reaction monitoring-mass spectrometric immunoassay responsive to parathyroid hormone and related variants

Mary F. Lopez, Taha Rezai, David A. Sarracino, Amol Prakash, Bryan Krastins, Michael Athanas, Ravinder Jit Singh, David R. Barnidge, Paul Oran, Chad Borges, Randall W. Nelson

Research output: Contribution to journalArticle

88 Citations (Scopus)

Abstract

BACKGROUND: Parathyroid hormone (PTH) assays able to distinguish between full-length PTH (PTH1-84) and N-terminally truncated PTH (PTH7-84) are of increasing significance in the accurate diagnosis of endocrine and osteological diseases. We describe the discovery of new N-terminal and C-terminalPTHvariants and the development of selected reaction monitoring (SRM)-based immunoassays specifically designed for the detection of full-length PTH [amino acid (aa)1-84] and 2 N-terminal variants, aa7-84 and aa34-84. METHODS: Preparation of mass spectrometric immunoassay pipettor tips and MALDI-TOF mass spectrometric analysis were carried out as previously described. We used novel software to develop SRM assays on a triple-quadrupole mass spectrometer. Heavy isotope-labeled versions of target peptides were used as internal standards. RESULTS: Top-down analysis of samples from healthy individuals and renal failure patients revealed numerous PTH variants, including previously unidentified aa28-84, aa48-84, aa34-77, aa37-77, and aa38-77. Quantitative SRM assays were developed for PTH1-84, PTH7-84, and variant aa34-84. Peptides exhibited linear responses (R2 = 0.90-0.99) relative to recombinant human PTH concentration limits of detection for intact PTH of 8 ng/L and limits of quantification of 16-31 ng/L depending on the peptide. Standard error of analysis for all triplicate measurements was 3%-12% for all peptides, with <5% chromatographic drift between replicates. The CVs of integrated areas under the curve for 54 separate measurements of heavy peptides were 5%-9%. CONCLUSIONS: Mass spectrometric immunoassays identified new clinical variants of PTH and provided a quantitative assay for these and previously identified forms of PTH.

Original languageEnglish (US)
Pages (from-to)281-290
Number of pages10
JournalClinical Chemistry
Volume56
Issue number2
DOIs
StatePublished - Feb 1 2010

Fingerprint

Parathyroid Hormone
Immunoassay
Monitoring
Assays
Peptides
Endocrine System Diseases
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Mass spectrometers
Isotopes
Area Under Curve
Renal Insufficiency
Limit of Detection
Software
Amino Acids

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

Lopez, M. F., Rezai, T., Sarracino, D. A., Prakash, A., Krastins, B., Athanas, M., ... Nelson, R. W. (2010). Selected reaction monitoring-mass spectrometric immunoassay responsive to parathyroid hormone and related variants. Clinical Chemistry, 56(2), 281-290. https://doi.org/10.1373/clinchem.2009.137323

Selected reaction monitoring-mass spectrometric immunoassay responsive to parathyroid hormone and related variants. / Lopez, Mary F.; Rezai, Taha; Sarracino, David A.; Prakash, Amol; Krastins, Bryan; Athanas, Michael; Singh, Ravinder Jit; Barnidge, David R.; Oran, Paul; Borges, Chad; Nelson, Randall W.

In: Clinical Chemistry, Vol. 56, No. 2, 01.02.2010, p. 281-290.

Research output: Contribution to journalArticle

Lopez, MF, Rezai, T, Sarracino, DA, Prakash, A, Krastins, B, Athanas, M, Singh, RJ, Barnidge, DR, Oran, P, Borges, C & Nelson, RW 2010, 'Selected reaction monitoring-mass spectrometric immunoassay responsive to parathyroid hormone and related variants', Clinical Chemistry, vol. 56, no. 2, pp. 281-290. https://doi.org/10.1373/clinchem.2009.137323
Lopez, Mary F. ; Rezai, Taha ; Sarracino, David A. ; Prakash, Amol ; Krastins, Bryan ; Athanas, Michael ; Singh, Ravinder Jit ; Barnidge, David R. ; Oran, Paul ; Borges, Chad ; Nelson, Randall W. / Selected reaction monitoring-mass spectrometric immunoassay responsive to parathyroid hormone and related variants. In: Clinical Chemistry. 2010 ; Vol. 56, No. 2. pp. 281-290.
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abstract = "BACKGROUND: Parathyroid hormone (PTH) assays able to distinguish between full-length PTH (PTH1-84) and N-terminally truncated PTH (PTH7-84) are of increasing significance in the accurate diagnosis of endocrine and osteological diseases. We describe the discovery of new N-terminal and C-terminalPTHvariants and the development of selected reaction monitoring (SRM)-based immunoassays specifically designed for the detection of full-length PTH [amino acid (aa)1-84] and 2 N-terminal variants, aa7-84 and aa34-84. METHODS: Preparation of mass spectrometric immunoassay pipettor tips and MALDI-TOF mass spectrometric analysis were carried out as previously described. We used novel software to develop SRM assays on a triple-quadrupole mass spectrometer. Heavy isotope-labeled versions of target peptides were used as internal standards. RESULTS: Top-down analysis of samples from healthy individuals and renal failure patients revealed numerous PTH variants, including previously unidentified aa28-84, aa48-84, aa34-77, aa37-77, and aa38-77. Quantitative SRM assays were developed for PTH1-84, PTH7-84, and variant aa34-84. Peptides exhibited linear responses (R2 = 0.90-0.99) relative to recombinant human PTH concentration limits of detection for intact PTH of 8 ng/L and limits of quantification of 16-31 ng/L depending on the peptide. Standard error of analysis for all triplicate measurements was 3{\%}-12{\%} for all peptides, with <5{\%} chromatographic drift between replicates. The CVs of integrated areas under the curve for 54 separate measurements of heavy peptides were 5{\%}-9{\%}. CONCLUSIONS: Mass spectrometric immunoassays identified new clinical variants of PTH and provided a quantitative assay for these and previously identified forms of PTH.",
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AU - Lopez, Mary F.

AU - Rezai, Taha

AU - Sarracino, David A.

AU - Prakash, Amol

AU - Krastins, Bryan

AU - Athanas, Michael

AU - Singh, Ravinder Jit

AU - Barnidge, David R.

AU - Oran, Paul

AU - Borges, Chad

AU - Nelson, Randall W.

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N2 - BACKGROUND: Parathyroid hormone (PTH) assays able to distinguish between full-length PTH (PTH1-84) and N-terminally truncated PTH (PTH7-84) are of increasing significance in the accurate diagnosis of endocrine and osteological diseases. We describe the discovery of new N-terminal and C-terminalPTHvariants and the development of selected reaction monitoring (SRM)-based immunoassays specifically designed for the detection of full-length PTH [amino acid (aa)1-84] and 2 N-terminal variants, aa7-84 and aa34-84. METHODS: Preparation of mass spectrometric immunoassay pipettor tips and MALDI-TOF mass spectrometric analysis were carried out as previously described. We used novel software to develop SRM assays on a triple-quadrupole mass spectrometer. Heavy isotope-labeled versions of target peptides were used as internal standards. RESULTS: Top-down analysis of samples from healthy individuals and renal failure patients revealed numerous PTH variants, including previously unidentified aa28-84, aa48-84, aa34-77, aa37-77, and aa38-77. Quantitative SRM assays were developed for PTH1-84, PTH7-84, and variant aa34-84. Peptides exhibited linear responses (R2 = 0.90-0.99) relative to recombinant human PTH concentration limits of detection for intact PTH of 8 ng/L and limits of quantification of 16-31 ng/L depending on the peptide. Standard error of analysis for all triplicate measurements was 3%-12% for all peptides, with <5% chromatographic drift between replicates. The CVs of integrated areas under the curve for 54 separate measurements of heavy peptides were 5%-9%. CONCLUSIONS: Mass spectrometric immunoassays identified new clinical variants of PTH and provided a quantitative assay for these and previously identified forms of PTH.

AB - BACKGROUND: Parathyroid hormone (PTH) assays able to distinguish between full-length PTH (PTH1-84) and N-terminally truncated PTH (PTH7-84) are of increasing significance in the accurate diagnosis of endocrine and osteological diseases. We describe the discovery of new N-terminal and C-terminalPTHvariants and the development of selected reaction monitoring (SRM)-based immunoassays specifically designed for the detection of full-length PTH [amino acid (aa)1-84] and 2 N-terminal variants, aa7-84 and aa34-84. METHODS: Preparation of mass spectrometric immunoassay pipettor tips and MALDI-TOF mass spectrometric analysis were carried out as previously described. We used novel software to develop SRM assays on a triple-quadrupole mass spectrometer. Heavy isotope-labeled versions of target peptides were used as internal standards. RESULTS: Top-down analysis of samples from healthy individuals and renal failure patients revealed numerous PTH variants, including previously unidentified aa28-84, aa48-84, aa34-77, aa37-77, and aa38-77. Quantitative SRM assays were developed for PTH1-84, PTH7-84, and variant aa34-84. Peptides exhibited linear responses (R2 = 0.90-0.99) relative to recombinant human PTH concentration limits of detection for intact PTH of 8 ng/L and limits of quantification of 16-31 ng/L depending on the peptide. Standard error of analysis for all triplicate measurements was 3%-12% for all peptides, with <5% chromatographic drift between replicates. The CVs of integrated areas under the curve for 54 separate measurements of heavy peptides were 5%-9%. CONCLUSIONS: Mass spectrometric immunoassays identified new clinical variants of PTH and provided a quantitative assay for these and previously identified forms of PTH.

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