Simultaneous Measurement of Collagen Cross-Link Markers and Advanced Glycation End-Products by Q-TOF LC/MS

Collagen is a major component of the extracellular matrix (ECM). Collagen cross-links play an important role in both physiological and pathological changes of collagen. It is theorized that cross-link markers change composition over time and play a role in collagen changes during aging, resulting in mechanical changes such as loss in elasticity, and decreased proteolytic susceptibility. Advanced glycation end-products (AGEs) are a type of senescent cross-link that arise as a result of protein modification due to exposure to sugar. Here, we use high performance liquid chromatography (HPLC) with mass spectrometry (MS) to identify cross-links in mouse heart, bovine meniscus and human meniscus samples to quantify collagen cross-links markers and AGEs simultaneously. LC separation was achieved using a Cogent Diamond Hydride column, a silica hydride column, and an aqueous normal phase chromatographic method. MS detection was performed on a Bruker maXis Q-TOF mass spectrometer operated in positive ion mode. Pyridinoline (PYR), deoxypyridinoline (DPD), dihydroxylysine-norleucine (DHLNL) and carboxymethyl-lysine (CML) were definitively identified in meniscus and/or heart samples using commercially available standards, and quantification of these compounds will be conducted in the near future. MS signals consistent with carboxymethyl-arginine (CMA), and carboxyethyl-lysine (CEL) were also found, but further testing is necessary to definitively validated this.