Collagen is the most abundant protein in animals and is a basic building block within almost every organ or tissue. It is an essential component of the extracellular matrix which provides strength and elasticity to tissues that possess a mechanical function such as bones, tendons, and cartilage. The collagen macro-structure is stabilised by covalent crosslinks and any alteration in their biosynthesis can lead to several diseases including bone fragility, skin hyperelasticity, and cardiovascular diseases. Upon ageing, collagen undergoes further modification by sugar metabolites resulting in the formation advanced glycation end products that are linked to the decreasing mechanical strength of collagen.<br/>The analysis of collagen crosslinks and advanced glycation end products has proven to be challenging due to their high hydrophilicity. They were historically analysed by cation exchange and reverse phase chromatography, methods not conducive to their simultaneous and rapid analysis in biological tissues. To overcome these problems, we developed a rapid method for the analysis of collagen crosslinks and advanced glycation end products using silica hydride column chromatography. Separation is achieved under 5 minutes using water and methanol or acetonitrile as the mobile phase,without any need for ion pairing agents or buffer salts. More importantly, the method is compatible with UV, fluorescence and mass spectrometry detection. Cross links in a number of different biological samples were analysed using this method and the results have led to an enhanced undersatanding of possible biosynthetic pathways and control mechanisms for the biosynthesis of these crosslinks, as well as the possibility of identifying new cross links.

Five years with silica hydride column: Analysis of collagen crosslinks and advanced glycation end products

ACS Spring Meeting 2021

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