Comparison of modification sites in glycated crystallin in vitro and in vivo

Abstract

Glycation of α-crystallin is responsible for age- and diabetic-related cataracts, which are the main cause of blindness worldwide. We optimized the method of identification of lysine residues prone to glycation using the combination of LC-MS, isotopic labeling, and modified synthetic peptide standards with the glycated lysine derivative (Fmoc-Lys(i,i-Fru,Boc)-OH). The in vitro glycation of bovine lens α-crystallin was conducted by optimized method with the equimolar mixture of [12C6]- and [13C6]d-glucose. The in vivo glycation was studied on human lens crystallin. The glycated protein was subjected to proteolysis and analyzed using LC-MS. The results of in vitro and in vivo glycation of α-crystallin reveal a different distribution of the modified lysine residues. More Amadori products were detected as a result of the in vitro reaction due to forced glycation conditions. The developed method allowed us to identify the glycation sites in crystallin from eye lenses obtained from patients suffering from the cataract. We identified K166 in the A chain and K166 in the B chain of α-crystallin as major glycation sites during the in vitro reaction. We found also two in vivo glycated lysine residues: K92 in the B chain and K166 in the A chain, which are known as locations for Amadori products. These modification sites were confirmed by the LC-MS experiment using two synthetic standards. This study demonstrates the applicability of the LC-MS methods combined with the isotopic labeling and synthetic peptide standards for analysis of post-translational modifications in the biological material.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-015-8487-7) contains supplementary material, which is available to authorized users.

Maillard Proteomics: Opening New Pages

“…Another informative labeling technique relies on incubation with [ 13 C 6 ]glucose under the conditions mimicking in vivo glycation [ 131 , 151 , 221 ]. In terms of this approach, relative quantification relied on doublet signals representing in vivo glycation with [ 12 C 6 ]glucose and in vitro incorporation of [ 13 C 6 ]glucose [ 131 , 151 , 250 ]. Finally, standard isotope dilution techniques might rely on synthetic 13 C, 15 N-labeled peptides, spiked to plasma samples for a high-throughput characterization of their glycated profiles by multiple reaction monitoring [ 189 ].…”

Non-enzymatic glycation of α-crystallin as an in vitro model for aging, diabetes and degenerative diseases

“…Simulation studies have shown that, although Trp 60 is exposed to solvent in the α-B subunit, it is buried in the dimer models, located at the interacting interface of the subunits (Guo 2008). Using LCMS and isotopic labeling, Kielmas et al (2015) have shown that K70, K88, K92, K99, K103, K145, K150 and K166 are the lysine glycation targets on α-crystallin. Gangadharaiah et al (2010) have reported that R12, R21, R54, R65, R69, R103, R112, R117, R119, R157 and R163 are arginine glycation sites on α-crystallin.…”

Arginine side-chain modification that occurs during copper-catalysed azide–alkyne click reactions resembles an advanced glycation end product

“…26 In a more recent study, several lysine modification sites were identified by LC-MS and confirmed by comparison with synthetic standards containing fructoselysine. 27 The chemical structures of many AGEs and their effects on protein structure and function, however, are still unknown. In this study, we identify a DHA modification on the side chain of arginine residues in peptides that occurs during CuAAC ligations in the presence of high concentrations of ascorbate.…”