Quantitative 'Immuno LC-MS/MS' of Clinically Relevant Heterogeneous Post Translational Protein Modifications: Oxidized and Truncated Parathyroid Hormone

摘要

Parathyroid hormone (PTH) plays a critical role in the regulation of circulating calcium levels, and serves as a biomarker of secondary hyperparathyroidism in the diagnosis of disorders such as vitamin D deficiency and chronic kidney disease (CKD), and for monitoring the effectiveness of treatment e.g., hemodialysis. Immunochemiluminescent assays for full length (i.e., 1-84) PTH have historically been problematic due to variable standardization, cross reactivity with heterogeneous truncated PTH fragments (e.g., 7-84), and variable correlations with biological parameters. A recent report that PTH may be oxidized in vivo at multiple sites further complicates these issues, since oxidized PTH is biologically inactive. To address these challenges, an immunocapture liquid chromatography-tandem mass spectrometry ('immuno LC-MS/MS') analysis strategy was developed and optimized for quantitative analysis of oxidized full length and truncated PTH isoforms. Importantly, a novel dual isotope-labeled internal standard approach (i.e., ~(15)N-labelled full length and truncated PTH and ~(13)C_6~(15)N_1 isotope-labelled oxidized PTH tryptic peptides added before and after extraction/sample handling) was used to quantitatively differentiate between in vivo and ex vivo oxidative modifications at methionine residues 8 and 18 in both full length and truncated PTH.