title: Selected reaction monitoring-mass spectrometric immunoassay responsive to parathyroid hormone and related variants.
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-terminal PTH variants 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 (R(2) = 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.
authors.affiliation: ThermoFisher Scientific BRIMS, Cambridge, MA, USA.
authors.forename: Mary F
authors.initials: MF
authors.lastname: Lopez
authors.forename: Taha
authors.initials: T
authors.lastname: Rezai
authors.forename: David A
authors.initials: DA
authors.lastname: Sarracino
authors.forename: Amol
authors.initials: A
authors.lastname: Prakash
authors.forename: Bryan
authors.initials: B
authors.lastname: Krastins
authors.forename: Michael
authors.initials: M
authors.lastname: Athanas
authors.forename: Ravinder J
authors.initials: RJ
authors.lastname: Singh
authors.forename: David R
authors.initials: DR
authors.lastname: Barnidge
authors.forename: Paul
authors.initials: P
authors.lastname: Oran
authors.forename: Chad
authors.initials: C
authors.lastname: Borges
authors.forename: Randall W
authors.initials: RW
authors.lastname: Nelson
mesh_terms: D000368:Aged; D000595:Amino Acid Sequence; D019540:Area Under Curve; D005260:Female; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007676:Kidney Failure, Chronic / Q000097:blood / Q000175:diagnosis*; D057230:Limit of Detection; D008297:Male; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000097:blood*; D010446:Peptide Fragments / Q000097:blood*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D010281:Parathyroid Hormone; D010446:Peptide Fragments; C414937:parathyroid hormone (7-84)