Enzymatic digestion and mass spectrometry in the study of advanced glycation end products/peptides

Abstract

An extensive study was carried out on HSA and non-enzymatically glycated HSA by enzymatic digestion with trypsin and endoproteinase Lys-C, with the aim of identifying specific glycated peptides deriving from enzymatic digestion of glycated HSA. They may be considered, in pectore, as advanced glycation end products/peptides. These compounds, important at a systemic level in diabetic and nephropathic subjects, are produced by enzymatic digestion of in vivo glycated proteins: They are related to the pathological state of patients and have been invoked as responsible for tissue modifications. The digested mixtures obtained by the two enzymes were analyzed by MALDI/MS and LC/ESI/MS n , and clear cut differences were found. First of all, the digestion products of glycated HSA are generally less abundant than those observed in the case of unglycated HSA, accounting for the lower proclivity of the former to enzymatic digestion. MS/MS experiments on doubly charged ions, comparisons with a protein database, and molecular modeling to identify the lysine NH 2 groups most exposed to glycation, identified some glycated peptides in digestion mixtures obtained from both types of enzymatic digestion. Residues 233 K, 276 K, 378 K, 545 K, and 525 K seem to be privileged glycation sites, in agreement with the fractional solvent accessible surface values calculated by molecular modeling. (J Am Soc Mass Spectrom 2004, 15, 496 -509)

Mass spectrometric characterization of glycated β-lactoglobulin peptides derived from galacto-oligosaccharides surviving the in vitro gastrointestinal digestion

“…Due to the complexity of the peptides profile and to the incomplete chromatographic separation between nonglycated and glycated peptides, analogous digestion patterns were apparently obtained for unglycated and glycated ␤-LG [13,19]. A similar behavior was observed for other proteins such as bovine serum albumin (BSA) [34] or HSA [14,38]. In good agreement with this, 33 of the 47 unglycated peptides identified in the digest of native ␤-LG were also detected in that of glycated ␤-LG (Table 2).…”

“…Although a large number of ions remained present in both samples, most of those present in the glycated ␤-LG were much less abundant. This fact could be attributed to a sum of multiple facts such as the lower ionization power of glycated peptides, the lower susceptibility to digestion for glycated proteins [14,34], or the partial glycation of Lys-containing and/or N-terminal peptides. Overall, 58 and 23 peptides covering 97% and 63% of the mature ␤-LG sequence could be identified by MALDI-MS in the digests of native and glycated samples, respectively ( Table 1).…”

“…Finally, nine new ionic species of low abundances were detected at m/z values of 1756. 8, 1567.6, 1113.5, 1097.5, 1059.4, 982.3, 976.3, 930.4, and 758.3. These mass values might correspond either to glycated short peptides or to unglycated peptides generated by new enzymatic cleavages as glycation may modify enzyme action [14]. Figure 3 illustrates the resulting total ion current (TIC) chromatogram for glycated and unglycated digested proteins.…”

“…The power of mass spectrometric techniques for the study of glycated and nonglycated peptides has been widely demonstrated [14,15]. Matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) have been used to determine the structure of proteins and peptides and their glycation products with monosaccharides [14,16,17].…”

“…Matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) have been used to determine the structure of proteins and peptides and their glycation products with monosaccharides [14,16,17]. In the case of ␤-LG, several articles describing the identification of lactose and galactose glycation sites using different mass spectrometric strategies have been published in the last decade [18 -24].…”