Quantitative and structural analysis of isotopically labelled natural crosslinks in type I skin collagen using LC-HRMS and SANS

Abstract

Collagen structure in biological tissues imparts its intrinsic physical properties by the formation of several covalent crosslinks. For the first time, two major crosslinks in the skin dihydroxylysinonorleucine (HLNL) and histidinohydroxymerodesmosine (HHMD), were isotopically labelled and then analysed by liquid-chromatography high-resolution accurate-mass mass spectrometry (LC-HRMS) and small-angle neutron scattering (SANS). The isotopic labelling followed by LC-HRMS confirmed the presence of one imino group in both HLNL and HHMD, making them more susceptible to degrade at low pH. The structural changes in collagen due to extreme changes in the pH and chrome tanning were highlighted by the SANS contrast variation between isotopic labelled and unlabelled crosslinks. This provided a better understanding of the interaction of natural crosslinks with the chromium sulphate in collagen suggesting that the development of a benign crosslinking method can help retain the intrinsic physical properties of the leather. This analytical method can also be applied to study artificial crosslinking in other collagenous tissues for biomedical applications.

Enzymatic and non-enzymatic crosslinks found in collagen and elastin and their chemical synthesis

“…103 The presence of HHMD 16 in vivo is debatable, despite it having been isolated, identified and quantified in different tissues by several researchers. 62,102,[120][121][122][123][124] Although both HHL 15 and HHMD 16 crosslinks were hypothesized to be laboratory artefacts, 113 these findings have been criticized. 114,115 Whether, HHL 15 and HHMD 16 are only artefacts found in mass-spectrometry, or are indeed a natural collagen crosslink remains uncertain.…”

“…This suggests that HHMD 16 is susceptible to degradation at low pH. 123 At this point no total organic synthesis of either of these crosslinks has been published to further understand the nature of these intriguing histidine-containing crosslinks.…”