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References of the abstract :
Introduction : The crosslinked carboxy-terminal telopeptide of type I collagen (B-CTX) is a biomarker used for the follow-up of osteoporotic patients. It is currently uncharacterized, making it impossible to standardise the various immunoassays for B-CTX. Little is known about the structure of the marker, other than the fact that it is a product of type I collagen digestion and consists of three peptides linked by a pyridinoline crosslink.
Material and methods : Plasma, serum, and urine pools were processed using two distinct sample preparation protocols. In the first sample preparation, protein precipitation was used to prepare and concentrate the pools. Using preparative liquid chromatography, samples were then separated, and fractions were collected every 15 seconds. Following evaporation, fractions were reconstituted with 30 uL of H2O, 0,4% FA. The other sample preparation consisted of affinity chromatography with two antibodies directed against a well-known portion of B-CTX. After samples were purified, they were evaporated and reconstituted with 30uL of injection solvent. Both protocols' samples were then injected into a high resolution mass spectrometer (Synapt XS, Waters). The mass spectra we obtained were then analyzed by PLGS and Peaks Studio for type I collagen peptides identification.
Results : We were able to identify ten uncrosslinked peptides of type I collagen containing the crosslink site so far but additional database searches are currently underway to identify crosslinked peptides. These findings would lead to a full characterization of B-CTX.
Conclusion : The successful development of our workflow led to the identification of several candidates. Confirmation of these candidates needs further investigation.
