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Chiral separation for proteomics in aging collagens

Résumé

Recent advances in separation sciences have allowed separating chiral compounds i.e. amino acids by LC-MS and CE-MS [1]. Naturally, proteins are found in their L-amino acid form. But, during the aging process, a natural amino acid racemization can take place. Indeed, D-amino acid detection in aging proteins has shown the correlation between the % D-amino acids and age [2]. Moreover, D-amino acids in aging proteins are liked to aging diseases. It is the case of D-Asp in several proteins linked to arteriosclerosis [2], Alzheimer’s disease [3], and cataracts [4]. For this work, the D-amino acid rates in recent and aging collagens, as the most abundant protein in animals including humans, were studied. Reverse-phase separation method by HPLC was used to separate and collect peptides, come from enzymatic digestion of collagen samples at different ages, on some fractions. Peptides in each fraction were separated and analyzed by reverse-phase nanoLC-MS to determine their exact position on the collagen sequences. Then, peptide fractions were hydrolyzed and derivatized, and proteinogenic amino acids enantiomers obtained including all common natural amino acids and modified amino acids such as hydroxyproline and methionine sulfoxide, were separated and analyzed by a chiral LC-MS method. In the first part, peptides obtained by enzymatic digestion and aging degradation were identified and compared according to sample ages. The number of peptides comes from aging degradation increases in old collagen samples. In the second part, the chiral amino acid separation has allowed to detect D-amino acids in collagen samples. The % D-amino acid in collagen samples was progressive in accordance with the age. Thanks to the sequence recovery made by the peptide analysis, the exact position of amino acids totally racemized was determined. Reverse-phase separation has allowed identifying peptides from aging degradation. The chiral separation method has allowed determining the D-amino acid proportions in collagen samples at different ages. The % D-amino acids – age correlation was established. For amino acids totally racemized on their D-form, their positions on the sequence were determined. This analysis strategy can also be applied to all proteins related to aging and/or diseases. This study will be the subject of a future publication. [1]M. Morvan and I. Mikšík, Recent Advances in Chiral Analysis of Proteins and Peptides, Separations, 2021, 8, 112. [2]J. T. Powell, N. Vine, and M. Crossman, On the accumulation of d-aspartate in elastin and other proteins of the ageing aorta, Atherosclerosis, 1992, 97, 201–208. [3]J. M. Lee, L. Petrucelli, G. Fisher, S. Ramdath, J. Castillo, M. M. Di Fiore, A. D’Aniello, Evidence for D-Aspartyl-β-Amyloid Secretase Activity in Human Brain, J Neuropathol Exp Neurol, 2002, 61, 125–131. [4]M. Y. S. Hooi and R. J. W. Truscott, Racemisation and human cataract. d-Ser, d-Asp/Asn and d-Thr are higher in the lifelong proteins of cataract lenses than in age-matched normal lenses, Age, 2011, 33, 131–141.
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Dates et versions

hal-04319101 , version 1 (02-12-2023)

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  • HAL Id : hal-04319101 , version 1

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Marine Morvan, Ivan Mikšík. Chiral separation for proteomics in aging collagens. MSB 2022, Jul 2022, Liège, Belgium. ⟨hal-04319101⟩
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