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.