Results
Separation of CTX species by preparative chromatography and
off-line 2D
LC/LC
Concentrated pools of serum and plasma, separated by preparative
chromatography, exhibited similar elution profiles regardless of the
kits used for quantitating the fractions, as illustrated in Figure 3.
However, there were significant variations in the concentrations
calculated by the different kits for highly concentrated fractions. This
discrepancy can potentially be attributed to the matrix effect, which
often has a substantial impact on immunoassays. Since our fractions
differ significantly from a biological matrix, it’s likely that the
various immunoassays are affected differently. In light of these
findings, it was determined that reliance on the IDS-iSYS CTX-I
(CrossLaps®) kit for further antibody-based quantitation would be more
appropriate.
Regarding retention time, CTX molecules appear to be highly hydrophilic,
given their elution at a very low percentage of ACN. The differences
observed in the elution profiles between urine and plasma/serum could be
attributed to modifications that CTX molecules undergo, such as
conjugation, in order to be excreted in urine. Consequently, different
CTX metabolites may be present in higher concentrations in urine.
Concerning the off-line 2D LC/LC separation, out of all the fractions
obtained during the initial step of preparative chromatography (figure
4A), nine (fractions 3, 5, and 17-23) displayed concentrations above 1
ng/mL, the arbitrarily chosen threshold, and were retained for the
second round of preparative chromatography (figure 4B). Two types of
elution profiles were observed for fractions 3, 5, 17-23. Fractions 5
and 17 displayed very similar profiles, both with two peaks at 1 minute
and 3.75 minutes, while the remaining fractions yielded elution profiles
with only one peak at different retention time. Given the similarity in
the elution profiles of fractions 5 and 17, it’s conceivable that the
species present in these fractions may be isomers. In the initial
chromatography, both isomers are separated into distinct fractions. It
is plausible that following post-column flow splitting, the isolated
isomer undergoes isomerization to transform into the other isomer,
reaching equilibrium in solution.
The fact that fractions 19 to 23 exhibited concentrations exceeding 1
ng/mL could be attributed to either significant tailing of the peak at
3.75 minutes or the presence of different molecules than those
responsible for the peak at 3.75 minutes. In light of these results, the
hypothesis that different CTX molecules coexist and are recognized by
the immunoassays gains credence, as the elution profiles obtained from
fractions 19 to 23 do not exhibit a peak at 3.75 minutes but rather at
4, 4.25, 4.5, 4.75, and 5 minutes.
Peptide identification
From the samples obtained after protein precipitation coupled with
preparative chromatography, a total of 502 linear peptides derived from
type I collagen were successfully identified. Among these, 22 peptides
containing the C-terminal pyridinoline crosslink site were identified.
Pyridinoline crosslinks involve the fusion of two telopeptide
hydroxyallysine and one helix lysine. While only hydroxylysine residues,
and by extension, hydroxyallysine, are directly implicated in
pyridinoline crosslinks at the telopeptide level, our focus also
encompasses peptides containing a lysine residue at the crosslink site
of the telopeptide. This broader consideration aims to gather
information about the cleavage sites around the crosslink site, rather
than identifying precursors of the crosslink. Peptides featuring
hydroxylation of proline residues (n=7) in the telopeptidic regions were
excluded, as this PTM is reported to be absent in this region of
collagen molecules [24,38–40]. Additional investigations are
required to validate the presence of hydroxyproline in the telopeptidic
peptides.
For the samples purified by affinity chromatography, 23 peptides
containing the epitope EKAHDGGR or EhylHDGGR were identified. These
peptides varied in length, ranging from 7 to 60 residues.
Hydroxyprolines were identified at various sites in 9 of these peptides.
However, no sugar-mediated PTMs were detected. It is worth noting that
the absence of glycosylation detection is not unexpected, as
glycosylation can be challenging to analyze using electrospray
ionization, the method employed in this study. Additionally, it is
crucial to acknowledge that certain PTMs, such as the oxidation of
hydroxylysine residues by lysyl oxidase—an essential step preceding
crosslinking—were not considered in this analysis. Consequently, more
linear peptides may have been present but not identified.
Significantly, no divalently crosslinked peptides were found in the
analysis. Divalent crosslinks are typically associated with
newly-synthetized bone tissue, serving as precursors to trivalent
crosslinks found in older bone tissue. The newly-synthetized bone tissue
rarely undergoes bone resorption compared to older bone tissue except in
some metabolic bone disorders, such as Paget’s disease. Therefore,
founding no divalently crosslinked peptides in the plasma and serum is
in line with the expectations.
All peptides containing a lysine or hydroxylysine residue involved in
pyridinoline crosslinks are detailed in Table 1.
Analysis of theorical trivalently crosslinked
models
Given the multitude of peaks observed in the chromatogram resulting from
affinity chromatography coupled to nano-LC-HR-MS, we postulated that the
number of crosslinked species was substantial. To achieve a
comprehensive identification of these species, particularly
acknowledging that trivalently crosslinked peptides cannot be directly
identified using standard peptide/protein identification software, we
developed model molecules. These model molecules consisted of three
peptides selected from the previously identified list of linear
peptides, crosslinked together by a pyridinoline or deoxypyridinoline.
Subsequently, these model molecules were employed in the chromatogram
search using specialized software.
A total of 3,230 model molecules, outlined in Supplemental Info 1, were
generated by combining the various peptides. The chemical formula for
each model molecule was calculated as part of this process.
Skyline was employed to identify traces corresponding to our model
molecules based on their chemical formulas. All model molecules were
successfully identified in samples obtained through preparative
chromatography (fractions 3, 5, 17-23) and samples obtained via affinity
chromatography in both plasma and serum.
For samples purified by preparative chromatography, the majority of the
molecules coeluted within the first 3 minutes of liquid chromatography.
Unfortunately, the peak shapes were poorly defined, characterized by low
intensity. This phenomenon could be attributed to the inherent
complexity of the sample, even after separation via preparative
chromatography. The important competition for ionization in the
ionization source becomes apparent when numerous molecules coelute.
Additionally, molecules like phospholipids and albumin, which
significantly compete for the signal, were not eliminated, further
contributing to this challenge.
In contrast, samples purified by affinity chromatography exhibited
chromatograms (figure 5) with fewer interferences and better peak
shapes, which can be attributed to the higher level of sample purity.
The peak intensities in these chromatograms were more than 50 times
higher.
Following Skyline analysis, similar chromatograms were obtained for both
plasma and serum using the same antibodies. However, commonalities were
identified among the four chromatograms, including the coelution of
numerous CTX species at 1.5 minutes and a prominent peak corresponding
to the ”3+3+4” species. While C-terminal pyridinoline and
deoxypyridinoline predominantly involve the α1 chain helix of type I
collagen and less the α2 chain helix, the ”3+3+4” species, composed of
two peptides, PQEKAHDGGR (α1 chain of type I collagen) crosslinked to
the peptide FKGIRGHNG (α2 chain of type I collagen) by a
deoxypyridinoline, was designated as the primary CTX species due to its
consistent presence in all chromatograms. It is important to note,
however, that this designation does not necessarily imply it is the most
concentrated species in the blood. The prominence of the “3+3+4”
species may stem from its superior ionization, possibly due to better
isolation from other molecules or its specific structure and
size[41,42].
Nonetheless, it is evident that different species were present in plasma
and serum, and distinct species were also captured by the antibodies. In
terms of differences between plasma and serum, it is apparent that
plasma contains larger species than serum. This distinction can be
explained by the fact that proteolysis occurs to a lesser extent in
plasma after collection, as plasma proteases are inhibited by the
chelation of divalent ions by EDTA. Moreover, it is evident that
degradation products were simultaneously captured with the initial
species they originated from, as very similar species were identified
within the same sample.
Regarding the differences between the species captured by the
antibodies, the most prevalent species differed for both antibodies.
There were no significant differences in terms of properties or
structures between the group of species recognized by antibody 1M0161
and the group of species recognized by antibody 1M0122, except that the
species captured by 1M0161 tended to have a lower retention time and
were thus more polar than the species captured by 1M0122.
However, a big number of the identified species were captured by the
same antibody in both matrices. Therefore, it can be hypothesized that
the capture of different species is reproducible.
Discussion
Osteoporosis, characterized by excessive bone resorption over bone
formation, diminishes bone mass, heightening fracture vulnerability.
Currently endorsed by the International Osteoporosis Foundation and the
International Federation of Clinical Chemistry and Laboratory Medicine
for the assessment of bone resorption in osteoporotic patients, CTX
faces challenges due to variable immunoassay outcomes, eroding clinician
confidence and limiting clinical use. Addressing this, the development
of a reliable reference method is imperative, but the incomplete
understanding of CTX structure hinders progress.
In this work, we employed a multi-step approach in order to fully
characterize CTX. We extracted CTX directly from human biological
matrices using preparative liquid chromatography (LC) and affinity
chromatography techniques. Preparative LC served as a straightforward
preparation step for the subsequent identification of uncrosslinked
linear peptides derived from type I collagen via high-resolution mass
spectrometry (HR-MS). The objective of the preparative
chromatography-based separation was to identify a comprehensive set of
peptides originating from type I collagen present in the blood,
independent of antibodies, in order to use them to build CTX models.
Affinity chromatography was then used for the analysis of CTX species,
as it provided a significantly more concentrated and purified sample
thanks to its high selectivity for CTX species. We then applied our
previously described proteomics workflow[36] to characterize all the
distinct CTX species present in both plasma and serum samples. This
marks a significant step towards enhancing our understanding of these
critical markers in the context of osteoporosis management.
The substantial number of CTX species identified in our study can be
attributed to the complex nature of CTX proteolysis, occurring at
multiple stages.
Firstly, our prior research on cathepsin K cleavage sites has
demonstrated that the digestion of type I collagen by cathepsin K
remains non-reproducible[36]. Consequently, during bone resorption,
multiple CTX species are produced. Moreover, once released into the
bloodstream, these species are susceptible to cleavage by circulating
metalloproteases and undergo further proteolysis in the liver by Kupffer
cells, leading to an increased number of coexisting species in the
bloodstream. Depending on the type of blood collection tube used,
proteolysis by circulating proteases may still occur after blood
collection.
In addition to cleavages, the presence of PTMs that are not uniform
further increases the variety of species observed in the chromatograms.
These PTMs primarily involve the hydroxylation of lysine and proline
residues, which are highly prevalent in type I collagen.
Given that an aspartic acid residue is located near the crosslink site,
many of the species may undergo isomerization resulting in the
manifestation of multiple species. However, β-isomerization of type I
collagen is associated with older bone tissue, suggesting that the
majority of resorbed bone tissue should exhibit β-isomerization.
Consequently, CTX should also predominantly exist in the β-isomeric
form, as it is released during bone resorption. Nevertheless, in
specific bone diseases such as Paget’s disease, α-CTX may be released
during bone resorption.
It is worth noting that smaller species were more prominent in the
chromatograms, possibly due to their higher abundance in the blood as a
result of in situ proteolysis. Additionally, larger molecules may ionize
less efficiently in the mass spectrometer source, and some of them may
have partially precipitated during the sample preparation process. The
observation that more large species were found in plasma aligns with
this theory.
A significant limitation in our study is the inability to doubly confirm
the presence of our model molecules through MS/MS spectra, given the
unknown and thus, unpredictable fragmentation pattern of such molecules.
Nevertheless, the superposition of traces from multiple states of charge
at the same retention time serves as an intriguing indicator of our
target, making it less likely to be an interference.
The diversity of CTX species identified and the differences in the
species recognized by the antibodies in the three commercially available
kits may contribute to the variations in results obtained by these
assays[43–45]. Each kit may use a different combination of
antibodies, leading to differential recognition of CTX species. However,
it’s worth noting that despite these differences, the overall results
from the kits do not show significant discrepancies. This suggests that
a significant portion of CTX species is still effectively captured by
the antibodies used in these assays, leading to relatively consistent
measurements across the kits.
In conclusion, this study successfully identified a substantial number
of CTX species extracted from human plasma and serum. Our findings
highlight the complexity of CTX proteolysis, and the diverse array of
species present in these biological matrices.
In light of our findings, it becomes evident that, prior to the
development of a LC-MS/MS method for the quantitation of all CTX
species, the development of a digestion step aimed at yielding a single,
standardized CTX species is crucial. Once this digestion step is
optimized within the biological matrix, we will be well-positioned to
progress towards the development and subsequent validation of an
LC-MS/MS method for the quantitation of the total CTX.
This method is anticipated to play a crucial role in advancing the
precision and standardization of CTX immunoassays, thereby improving
osteoporosis monitoring. Following standardization, these immunoassays
are expected to exhibit reduced variability, becoming fully
interchangeable. This, in turn, is projected to instill greater
confidence among clinicians, fostering enhanced utilization of the
marker in patient follow-up. With improved assessment of medication
compliance, the risk of fractures is anticipated to decrease in
osteoporotic patients.