Figure 1. Developed workflow. Plasma and serum samples, prepared through protein precipitation, underwent separation via LC preparative. The fractions obtained were then subjected to CTX concentration analysis. Fractions containing CTX concentrations exceeding 1 ng/mL were subsequently analyzed using HR-MS. The acquired spectra were processed using PEAKS software for the identification of linear peptides derived from type I collagen. Peptides that contained lysine residues involved in pyridinoline crosslinks were selected. These selected peptides were then employed in the construction of trivalently crosslinked model molecules. The primary purpose of these models was to comprehensively represent all potential CTX species. In parallel, plasma and serum samples were purified through affinity chromatography and likewise analyzed using HR-MS. The spectra obtained were also processed using PEAKS to identify linear peptides, which were used to construct additional trivalently crosslinked model molecules. Once all the model molecules were designed, Skyline software was employed to identify trivalently crosslinked CTX species.

Figure 2. Off-line 2D preparative LC/LC separation protocol. A concentrated urine sample was prepared and concentrated using protein precipitation. This prepared sample was then subjected to separation through preparative LC. The fractions obtained from this separation were subsequently assessed for their CTX concentration, and an elution profile was established.

Fractions with CTX concentrations exceeding 1 ng/mL were singled out for a second round of preparative LC. The fractions from each of these selected fractions, which exceeded 1 ng/mL, were subjected to a second run of preparative LC. The results obtained from this process were compiled into a comprehensive elution profile.

Figure 3. Elution profile obtained after preparative chromatography separation of human plasma and serum. Fractions levels of CTX were assessed using the iSYS CTX-I (CrossLaps®) and CrossLaps® ELISA kits from IDS Diagnostics, as well as the Β-Crosslaps ECLIA kit by Cobas (Roche).

Figure 4. Elution profile obtained by off-line 2D preparative LC/LC separation of concentrated urine samples. A) First run of preparative LC B) second run of preparative LC.

Figure 5. Chromatograms obtained from a) Plasma sample captured by antibody 1M0161, b) Plasma sample captured by antibody 1M0122, c) Serum sample captured by antibody 1M0161 and d) Serum samples captured by antibody 1M0122

Supplemental info 1. Schematic representation of all model molecule.