Analysis of the Site-Specific Myoglobin Modifications in the Melibiose-Derived Novel Advanced Glycation End-Product

Abstract

MAGE (melibiose-derived advanced glycation end-product) is the glycation product generated in the reaction of a model protein with melibiose. The in vivo analog accumulates in several tissues; however, its origin still needs explanation. In vitro MAGE is efficiently generated under dry conditions in contrast to the reaction carried in an aqueous solvent. Using liquid chromatography coupled with mass spectrometry, we analyzed the physicochemical properties and structures of myoglobin glycated with melibiose under different conditions. The targeted peptide analysis identified structurally different AGEs, including crosslinking and non-crosslinking modifications associated with lysine, arginine, and histidine residues. Glycation in a dry state was more efficient in the formation of structures containing an intact melibiose moiety (21.9%) compared to glycation under aqueous conditions (15.6%). The difference was reflected in characteristic fluorescence that results from protein structural changes and impact on a heme group of the model myoglobin protein. Finally, our results suggest that the formation of in vitro MAGE adduct is initiated by coupling melibiose to a model myoglobin protein. It is confirmed by the identification of intact melibiose moieties. The intermediate glycation product can further rearrange towards more advanced structures, including cross-links. This process can contribute to a pool of AGEs accumulating locally in vivo and affecting tissue biology.

Identification of Heavily Glycated Proteoforms by Hydrophilic-Interaction Liquid Chromatography and Native Size-Exclusion Chromatography – High-Resolution Mass Spectrometry

“…To date, many AGEs have been described (Figure S2), such as Nε-carboxymethyl-lysine (CML), Nε-(1-carboxyethyl)-lysine (CEL), Nε-(5-hydro-4imidazolon-2-yl)-ornithine (GH), methylglyoxal-lysine dimer (MOLD), etc. [46,47]. A comprehensive understanding of these AGEs plays an important role in understanding the processes of disease development, but currently, methods to test this at the intact level are lacking.…”

Analysis of N‐ and O‐linked site‐specific glycosylation by ion mobility mass spectrometry: State of the art and future directions

“…linked to specific sites on glycoproteins. By employing mass spectrometry (MS) in glycoproteomics, researchers have been able to profile glycoprotein populations and uncover valuable insights into glycan biomarkers, glycosylation changes associated with health and disease, and the functional roles of glycoproteins in various biological processes [21,24,55,[57][58][59][60][61][62][63][64].…”