Introduction
Osteoporosis is a skeletal disease characterized by low bone mass and
loss of bone microarchitecture, which represents a major global health
issue [1–3]. It accounts for more than 4% of the annual EU health
expenditure[2–4]. The disease is caused by an increased rate of
bone resorption, which involves osteoclasts releasing acids and
proteases like cathepsin K to digest type I collagen.[5] This
process results in the release of collagen-derived peptides into the
bloodstream. Due to the importance of finding biomarkers for
osteoporosis and other bone metabolism disorders, these peptides have
been extensively studied and investigated as potential
biomarkers[6–11].
During the course of these investigations, the primary emphasis was put
on the analysis of pyridinoline/deoxypyridinoline crosslinks, highly
specific to collagen molecules, and peptides bound to these crosslinks.
This particular focus was intentional, as the preference for these
markers over linear peptides stemmed from the fact that pyridinoline
crosslinks present in biofluids are primarily derived from bones due to
bone resorption [12]. This specificity makes them indicative of bone
metabolism, whereas linear peptides could originate from various tissues
rich in type I collagen. Furthermore, the inclusion of deoxypyridinoline
crosslinks in the study was driven by their specificity to bones and
dentin.[13,14] The outcomes of these investigations led to the
identification and characterization of trivalently crosslinked
C-telopeptide of the α1-chain of type I collagen (ICTP) and free
pyridinoline[15–24]. These characterizations relied on preparative
chromatography coupled with UV detection, MALDI TOF spectrometry, and
N-sequencing analysis[25–27]. Subsequently, immunoassays were
developed for the quantitative analysis of ICTP[28,29].
While the trivalently crosslinked C-Terminal telopeptide (CTX),
trivalently crosslinked N-Terminal telopeptide (NTX), and amino-terminal
propeptide of type I procollagen (PINP) can presently be quantified
through immunoassays, their comprehensive characterization remains
incomplete. Only specific portions of these molecules, such as the
sequence EKAHDGGR in CTX, are recognized and employed as epitopes in
immunoassays[28,30,31]. CTX and NTX are known to be made up of three
peptides trivalently crosslinked together by a pyridinoline
crosslink[32]. CTX and ICTP both originate from the same type I
collagen region and share a common origin. However, they are released
under different conditions and are cleaved by distinct enzymes.
Specifically, CTX is released through the activity of cathepsin K during
physiological bone resorption, while ICTP is likely cleaved by matrix
metalloproteases during pathological bone resorption, as observed in
bone metastasis[33].
To this day, CTX and PINP serve as the recommended markers for bone
resorption and bone formation, respectively, according to the guidelines
set forth by the International Osteoporosis Foundation and the
International Federation of Clinical Chemistry and Laboratory Medicine
for monitoring the therapeutic progress of osteoporotic
patients[34,35]. Despite CTX endorsement, the absence of a reference
method and variability in immunoassay results have diminished clinician
confidence, leading to underutilization. The lack of complete marker
characterization hampers the development of a definitive reference
method, like liquid chromatography coupled to tandem mass spectrometry
(LC-MS/MS), for precise quantitation. HR-MS analysis. Consequently, our
research is dedicated to the comprehensive characterization of CTX found
in human plasma and serum. Developing a LC-MS/MS method necessitates
precise knowledge of the exact structure of the targeted molecule. This
is crucial to achieve the highest level of specificity attainable by
triple quadrupole mass spectrometry. In contrast to the development of
immunoassays, where specific regions of a molecule can be targeted,
LC-MS/MS methods require the comprehensive targeting of the entire
molecule. This is because the method relies on the measurement of both
the mass and charge of the molecule, demanding a comprehensive
understanding of its structure. Therefore, we conducted an analysis of
the various CTX species present in plasma and serum using a
comprehensive workflow involving preparative chromatography, affinity
chromatography, and HR-MS analysis.