Collagen is the most abundant protein in animals and the major fibrous protein in connective tissues including skin, bone and tendon. The collagen molecule has a triple-helical structure stabilised by covalent crosslinks which are biosynthesised by enzymatic and non-enzymatic pathways. The formation of enzymatic crosslinks is initiated by lysyl oxidase, and the non-enzymatic known as advanced glycation end products are derived from the adventitious crosslinking reaction between carbohydrates and amino acid residues. Enzymatic and non-enzymatic crosslinks have previously been analysed by cation exchange and reverse phase chromatography. However, these methods cannot offer simultaneous analysis in connective tissues. Although few analytical methods for simultaneous determination of several collagen crosslinks have been developed, there are limitations to the quantitative and qualitative analyses of both enzymatic and non-enzymatic crosslinks. In this study, we have developed a rapid and accurate method for the analysis of enzymatic and non-enzymatic collagen crosslinks in skin. The separation was achieved in under 10 minutes on the silica hydride column using water and methanol/acetonitrile as mobiles phases. The eluted crosslinks were detected using high-resolution accurate mass spectrometry with a sub-pmol sensitivity.<br/>Additionally, the separation method on silica hydride column is suitable for UV, fluorescence and mass spectrometry detection. The feasibility of running MS detection in conjunction with UV and fluorescence detection which are very commonly used detectors for HPLC analysis as well as for preparative work was demonstrated. The method was applied to different animal skins for collagen crosslink analysis which is essential to understand the possible biosynthetic pathways and control mechanism of enzymatic and non-enzymatic collagen crosslinks.

Rapid and quantitative analysis of collagen crosslinks in skins using a silica hydride column and mass spectrometry detection

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.