With an aging population, the increased interest in the monitoring of skeletal diseases such as osteoporosis led to significant progress in the discovery and measurement of bone turnover biomarkers since the 2000s. Multiple markers derived from type I collagen, such as CTX, NTX, PINP, and ICTP, have been developed. Extensive efforts have been devoted to characterizing these molecules
however, their complex crosslinked structures have posed significant analytical challenges, and to date, these biomarkers remain poorly characterized. Previous attempts at characterization involved gel-based separation methods and MALDI-TOF analysis on collagen peptides directly extracted from bone. However, using bone powder, which is rich in collagen, does not represent the true structure of the peptides in the biofluids as it was cleaved. In this study, our goal was to characterize plasma and serum CTX for subsequent LC-MS/MS method development. We extracted and characterized type I collagen peptides directly from human plasma and serum using a proteomics workflow that integrates preparative LC, affinity chromatography, and HR-MS. Subsequently, we successfully identified numerous CTX species, providing valuable insights into the characterization of these crucial biomarkers.