Selected reaction monitoring-mass spectrometric immunoassay responsive to parathyroid hormone and related variants.
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.
Lopez
Mary F
MF
ThermoFisher Scientific BRIMS, Cambridge, MA, USA.
Rezai
Taha
T
Sarracino
David A
DA
Prakash
Amol
A
Krastins
Bryan
B
Athanas
Michael
M
Singh
Ravinder J
RJ
Barnidge
David R
DR
Oran
Paul
P
Borges
Chad
C
Nelson
Randall W
RW
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*
D010281:Parathyroid Hormone; D010446:Peptide Fragments; C414937:parathyroid hormone (7-84)