|Mary F Lopez, ThermoFisher Scientific BRIMS|
Taha Rezai, ThermoFisher Scientific BRIMS
Bryan Krastins, ThermoFisher Scientific BRIMS
Amol Prakash, ThermoFisher Scientific BRIMS
David A Sarracino,ThermoFisher Scientific BRIMS
Michael Athanas, VAST Scientific, 790 Memorial Dr., Cambridge, MA 02139
Ravinder J Singh, Mayo Clinic College of Medicine, Rochester, MN 55905
David R Barnidge, Mayo Clinic College of Medicine, Rochester, MN 55905
Paul Oran, Biodesign Institute, Arizona State University, Tempe, AZ 85287
Chad Borges, Biodesign Institute, Arizona State University, Tempe, AZ 85287
Randall W Nelson, Intrinsic Bioprobes, Tempe, AZ 85287
|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. However, there are indications that greater microheterogeneity exists within PTH, which has yet to be fully characterized to determine the potential clinical utility and/or confounding effects it may have on present-day assays. The accurate examination of known PTH variants, while simultaneously evaluating other possible variants, requires a degree of analytical freedom that universally escapes conventional assays. Here we describe mass spectrometric immunoassays that while specifically tooled for the detection of PTH1-84 and PTH7-84, are able to simultaneously discover and evaluate unforeseen consequences of microheterogeneity in PTH.|
Mass spectrometry-based selective reaction monitoring assays (SRM) are rapidly becoming a preferred technology for the development of quantitative protein or peptide assays for clinical research. SRM assays deliver high sensitivity, selectivity and throughput, and when taken together, these parameters provide a breakthrough quantification methodology. During LC MS/MS, multiple charge states and fragmentation ions are generated from each fragment, resulting in upward of 1,000 different parent/daughter transitions possible for the digested PTH. Due to time and cost restrictions, the empirical investigation of each transition is not efficient. Therefore, a workflow was developed that incorporated predictive algorithms, utilization of previously acquired high-resolution MS trap-based discovery data and iterative optimization. This workflow was applied to predict and refine the optimal transitions for monitoring of target peptides, including peptides for routine monitoring of full length PTH (aa1-84) and peptides specific for clinically observed variants (aa7-84, 34-84). The strategy facilitates the translation of empirically obtained peptide intensity and fragmentation behavior from high resolution LC-MS/MS to triple quadrupole MS SRM assays. Inherent to the success of the workflow is the similarity of peptide ion fragmentation behavior in trap and triple quadrupole instrument.
Using this approach, an initial list of transitions was generated and queried empirically to produce an LC-MS/MS profile based on four target peptides that collectively spanned > 50 % of the full-length PTH sequence (45 of 84 amino acids). In addition, transitions for two peptides, were added to the profile to monitor for truncated variants PTH7-84 and PTH34-84, respectively. The optimized SRM assay was then used to interrogate a small cohort of clinical patient samples.