Generation and Characterization of a Protein Calibrator for Collagen Turnover Assays

Abstract

Background: Despite being banned by most international sports leagues and the International Olympic Committee, the widespread use of GH as a performance-enhancing drug is recognized by the athletic community. Illicit use of GH by competitors compromises both the safety of the individual as well as the perception of fair play and fair competition. Current methods for the detection of GH abuse include several commercial immunoassays to quantify P3NP in serum, which detect the downstream effects of GH abuse. Importantly, when analyzing the same set of samples, these immunoassays provide disparate results demonstrating significant bias. As a result, we aim to generate distributable reference materials that can be used to address these analytical challenges.Methods: T3PC produced by fibroblasts cell culture was isolated by multiple precipitation steps, followed by either size exclusion or anion exchange chromatography. The purity and concentration of this material was characterized using SDS-PAGE and LC-MS/MS analysis utilizing stable isotope labelled internal standard peptides. Results: A highly pure preparation of T3PC was not obtained. Based SDS-PAGE analysis, the intact form of T3PC was estimated to represent approximately 30.4% of the total material, with an estimated concentration of the family of proteins that contain pT1 (i.e., P3NP) in the purified calibrator is 1.226 μM. Conclusions: This material will be used to value-assign the family of T3PC proteins in a pooled human serum sample, which will subsequently be distributed to anti-doping laboratories worldwide to support global platform harmonization. Further purification of the material will be necessary to advance metrological standardization.