Materials and methods
Proteins precipitation
One pool of plasma and one of serum were created by combining leftover
plasma and serum samples from hemodialyzed patients, without considering
distinctions based on gender, health, or age. Such samples were chosen
to maximize peptide concentration since the peptides are cleared by the
kidney. To precipitate the proteins in these pools, 250 µL of H2O and
10% ZnSO4 (w/v) per milliliter of the matrix were employed. All
solutions were prepared using LC-MS grade solvents. The pools were
subjected to agitation on a reciprocating shaker for 20 minutes at 10°C
and subsequently centrifuged at high speed for 10 minutes. The resulting
supernatant was transferred to 2mL LoBind Eppendorf tubes and then
evaporated to dryness overnight under vacuum conditions at 35°C.
Following evaporation, the pools were reconstituted with 1mL of a
solution containing H2O, 5% dimethyl sulfoxide (DMSO),
and 0.4% formic acid (FA). The FA and DMSO were procured from
Sigma-Aldrich (Saint-Louis, MO, US).
Preparative
chromatography
A total of 50 µL of the concentrated pool was introduced into a Shimadzu
Nexera X2 UPLC system (Shimadzu Corporation, Kyoto, Japan).
Chromatographic separation was achieved using a XSelect PRM PST HSS T3
column (Waters) with a particle size of 2.5µm and dimensions of
2.1x150mm, maintained at 35°C. The mobile phases consisted of
H2O and acetonitrile (ACN) with the addition of 5% DMSO
and 0.4% FA. A gradient method was employed at a flow rate of 0.5
mL/min, as follows: initiation and maintenance at 0% of phase B (ACN,
5% DMSO, 0.4% FA) for 0.5 minute, followed by an increase to 10% of
phase B over 9 minutes, then a 1-minute period at 100% of phase B, and
finally a 5-minute step at 0% of phase B. Post-column flow splitting
was conducted every 15 seconds. Fractions obtained were subsequently
evaporated and reconstituted with 30 µL of H2O
containing 5% DMSO and 0.4% FA. The retention times of CTX were
determined by quantifying CTX in fractions collected through post-column
flow splitting during the entire run, utilizing the IDS-iSYS CTX-I
(CrossLaps®) and CrossLaps® ELISA kits from Immunodiagnostic System
(IDS), as well as the Β-Crosslaps ECLIA kit by Cobas (Roche).
Off-line 2D LC/LC
We employed an off-line 2D LC/LC technique to gain deeper insights into
the nature of the peaks observed in the elution profile obtained through
preparative liquid chromatography. Given the presence of multiple peaks,
it was imperative to determine whether these peaks resulted from
suboptimal LC parameter optimization or if they represented distinct
species.
Due to its elevated CTX concentration, urine was selected as the primary
human matrix for 2D LC/LC analysis. A total of 50 µL of urine
concentrated through evaporation was subjected to separation utilizing
the previously described method. Fractions obtained were assessed for
their CTX content using the IDS-iSYS CTX-I (CrossLaps®) kits by IDS.
Fractions exhibiting a high CTX concentration were subsequently
evaporated to dryness overnight under vacuum conditions at 35°C and
reconstituted with 100 µL of the injection solvent. Each reconstituted
fraction was then subjected to separation once more using the same
preparative liquid chromatography method as previously explained. The
CTX content of each resulting fraction was assessed using the IDS-iSYS
CTX-I (CrossLaps®) kit. Off-line 2D preparative LC/LC separation
workflow is represented in figure 1.
Affinity chromatography
Antibodies specific to β-CTX were generously provided by IDS (Boldon,
UK). These antibodies, named 1M0161 and 1M0122, target a
well-established β-CTX sequence: EKAHDGGR. Both 1M0161 and 1M0122
antibodies are integral components of the commercially available kits
for Β-CTX quantitation.
Regarding the columns used for affinity chromatography, the packing
process was conducted in-house. For the 1M0122 column, a rProtein A
Sepharose™ Fast Flow column (Merck) was initially
equilibrated with PBS. Subsequently, 12 mg of 1M0122 antibodies,
pre-diluted in PBS, were introduced into the column. Any excess
antibodies were removed by washing with PBS before injecting 35 mL of
pooled plasma/serum. The flow-through was collected, and immune
complexes were subsequently eluted using a solution of
H2O and citrate at pH 3.
For the 1M0161 column, a pre-activated NHS column was packed and stored
in 7 mL of isopropanol. After equilibrating the column with
H2O containing 1 mM HCl, 11.3 mg of 1M0161 antibodies,
diluted in a solution of H2O and NaHCO3at 100 mM, were injected twice. Unbound antibodies were washed with a
carbonate buffer. To saturate any remaining free NHS groups, a solution
of H2O, Ethanolamine at 0.5M, NaCl at 0.5M, and pH 8.30
was injected into the column (70 mL). The column was subsequently washed
and conditioned twice using the following solutions in the specified
order: H2O, citrate at 0.1M, NaCl at 0.5M, and pH 4;
H2O, NaCl at 1M, glycerol at 5%, KI at 0.1 mM, Triton X
100 at 0.1%, and NH4OH at 0.05%; H2O,
ethanolamine at 0.5M, NaCl at 0.5M, and pH 8.30. Finally, the column was
equilibrated in PBS before injecting 35 mL of pooled plasma/serum. The
flow-through was collected, and the antigens were eluted using a
solution of H2O and glycine at 0.1M and pH 3.
Subsequently, the samples were evaporated and reconstituted in
H2O containing 0.4% FA before injection.
Nano-LC-DIA analysis
Samples obtained following preparative chromatography and affinity
chromatography were introduced into the nanoAQUITY UPLC-system Nano-LC
(Waters), which was coupled to the SYNAPT XS mass spectrometer (Waters).
The column employed for chromatographic separation was the nanoEaseTM
M/Z HSS T3 column with a pore size of 100Å, particle size of 1.8µm, and
dimensions of 300µm x 150mm (Waters). The mobile phases consisted of a
mixture of H2O and ACN, supplemented with 0.1% formic
acid (FA). The gradient began by holding at 0% of ACN for 2 minutes and
subsequently increased to 90% over a span of 43 minutes, with a total
flow rate of 5 µL/min.
Data acquisition was conducted according the parameters described
previously in our previous work[36]. Data-independent analysis (DIA)
was chosen as acquisition mode in order to maximize peptide
identification.
Data processing
Type I collagen-derived linear peptides and their post-translational
modifications (PTMs) were identified using PEAKS X software
(Bioinformatics Solutions Inc., Waterloo, CA). Sequences of type I
collagen α1 and type I collagen α2 were obtained from the UniProt
database (https://www.uniprot.org/) and utilized for data-based
research. Variable PTMs, including hydroxylation of lysine and proline,
oxidation of methionine, glycation, and glycosylation, were considered.
Hydroxylation is a common occurrence in type I collagen, and oxidation
can potentially occur during sample preparation. Glycation and
glycosylation are also known to be present in type I collagen. For
in-silico digestion performed by PEAKS X Software, the settings were
configured as ”unspecific” since various enzymes, such as matrix
metalloproteinases (MMPs), may cleave CTX, especially in the
bloodstream, subsequent to its initial cleavage by cathepsin K.
The identification of divalently crosslinked peptides was carried out
using Stavrox/Merox software, as described by Götze et al . in
2012[37], and accessible at http://stavrox.com/Download.htm .
To identify trivalently crosslinked peptides, their chemical formulas
were determined and visualization of chromatogram was done using Skyline
software, available at https://skyline.ms/project/home/begin.view.
Confirmation of the presence of model
molecules in high resolution-MS spectra
The MS1 data obtained during UDMSE analysis were
extracted and subjected to analysis using Skyline to detect the presence
of model molecules. These model molecules were designed by appropriately
combining the identified peptides that contained lysines and
hydroxylysines involved in the C-terminal pyridinoline crosslink. The
entire workflow is depicted in Figure 2.