Abstract Title

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

Additional Funding Sources

The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant No. P20GM103408 and COBRE in Matrix Biology.

Abstract

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.

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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.