Isolation and characterization of collagen type I crosslink from skin: high-resolution NMR reveals diastereomers of hydroxylysinonorleucine crosslink
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Skin is made up of mainly collagen type I and its structure is stabilised by the formation of covalent immature and mature crosslinks. In this study, collagen immature crosslink hydroxylysinonorleucine (HLNL) was isolated from bovine skin in high purity using two sequential purification steps. These consisted of preparative fibrous cellulose and size exclusion chromatography. The purified crosslink was then analysed using tandem mass spectrometry and high-resolution nuclear magnetic resonance (NMR) spectroscopy. The mass of singly and doubly charged ions of HLNL was 292.1865 and 146.5970 m/z and their optimised fragmentation energy was 17 keV and 5 keV, respectively. The 13C NMR of HLNL showed a doubled-up peak at 67.84 and 67.91 ppm which corroborated a diastereomeric form of collagen immature crosslink HLNL and both are chiroptically indistinguishable. The chemical structure was fully resolved using 1H, 13C and DEPT-135 high-resolution NMR spectroscopy and compared with other previous studies. We also obtained for the first time the 2D NMR spectra COSY and HSQC of HLNL. We therefore suggested that collagen organization into specific fibrils’ orientation may be affected by the different configuration of these diastereomers of HLNL.
KeywordsSkin Collagen type I Unusual amino acids Preparative chromatography NMR Hydroxylysinonorleucine crosslink
Distortionless enhancement by polarization transfer-135
Heteronuclear single quantum coherence
Nuclear magnetic resonance
Total ion chromatogram
Thin layer chromatography
This work was supported by NZ Leather and Shoe Research Association (LASRA®), Palmerston North, New Zealand through the Ministry of Business, Innovation and Employment (MBIE) grant number LSRX1301 and LSRX1801.
The authors’ contributions to the manuscript were as follows: RN designed the research; RN, GN and PE conducted the research; and RN and GN wrote the paper. All authors have read and approved the final manuscript.
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Conflict of interest
The authors declare that they have no conflict of interest financial or non-financial.
Research involving human participants and/or animals
No human participants or animal model were involved with this project, thus exempting this study from animal/human approval process.
No patient samples or bio-bank samples were used for this study, thus exempting informed consent requirement. We confirm that this work is new and original and that the manuscript has not been published elsewhere, nor is it under consideration by any other journal. Furthermore, all the authors listed contributed in some way to the work.
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