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.