Glycation Isotopic Labeling with C-Reducing Sugars for Quantitative Analysis of Glycated Proteins in Human Plasma*□S

Non-enzymatic glycation of proteins is a post-translational modification produced by a reaction between reducing sugars and amino groups located in lysine and arginine residues or in the N-terminal position. This modification plays a relevant role in medicine and food industry. In the clinical field, this undesired role is directly linked to blood glucose concentration and therefore to pathological conditions derived from hyperglycemia (>11 mM glucose) such as diabetes mellitus or renal failure. An approach for qualitative and quantitative analysis of glycated proteins is here proposed to achieve the three information levels for their complete characterization. These are: 1) identification of glycated proteins, 2) elucidation of sugar attachment sites, and 3) quantitative analysis to compare glycemic states. Qualitative analysis was carried out by tandem mass spectrometry after endoproteinase Glu-C digestion and boronate affinity chromatography for isolation of glycated peptides. For this purpose, two MS operational modes were used: higher energy collisional dissociation-MS2 and CID-MS3 by neutral loss scan monitoring of two selective neutral losses (162.05 and 84.04 Da for the glucose cleavage and an intermediate rearrangement of the glucose moiety). On the other hand, quantitative analysis was based on labeling of proteins with [C6]glucose incubation to evaluate the native glycated proteins labeled with [C6]glucose. As glycation is chemoselective, it is exclusively occurring in potential targets for in vivo modifications. This approach, named glycation isotopic labeling, enabled differentiation of glycated peptides labeled with both isotopic forms resulting from enzymatic digestion by mass spectrometry (6-Da mass shift/glycation site). The strategy was then applied to a reference plasma sample, revealing the detection of 50 glycated proteins and 161 sugar attachment positions with identification of preferential glycation sites for each protein. A predictive approach was also tested to detect potential glycation sites under high glucose concentration. Molecular & Cellular Proteomics 9:579–592, 2010. Among post-translational modifications (PTMs) of proteins, non-enzymatic glycation is one of the less frequently studied in proteomics. Glycated proteins are formed by a non-enzymatic reaction between reducing carbohydrates (e.g. glucose, fructose, ribose, or derivatives such as ascorbic acid) with amino groups located in the N-terminal position or in lysine and arginine residues. It is worth emphasizing the differences between glycation and glycosylation. The latter is enzymatically catalyzed by glycosyltransferase and occurs in specific protein side chains such as asparagine (N-linked), serine and threonine (O-linked), and the C termini of cell surface proteins (1). Glycosylation is involved in many biological processes in contrast to glycation, which is a completely undesired modification from a clinical point of view. Because of the crucial role of glucose as an energy source in humans, it is the main circulating sugar and thus the most relevant molecule in terms of protein glycation. The mechanisms involved in glycation are illustrated in Fig. 1 for glucose as the reducing sugar (2). The process starts with the formation of the Schiff base by a condensation reaction between the carbonyl group of the reducing sugar and the amino group of the protein. The next step is the conversion of the thermodynamically unstable Schiff base into the Amadori compound that is considered as the first glycation level. Finally, the Amadori compound undergoes a series of dehydration and fragmentation reactions, generating a variety of carbonyl compounds such as methylglyoxal, glyoxal, glucosones, deoxyglucosones, and dehydroascorbate (3). These carbonyl compounds are generally more reactive than the original carbohydrate and act as propagators by reactions with free amino groups, leading to the formation of a variety of heterogeneous structures irreversibly formed and commonly known as advanced glycation end products. The impact of glycation encompasses alterations of the structure, function, and turnover of proteins (4). Evidently, the effects on biological function will depend on the extent of glycation. From a clinical point of view, the detection of this PTM at the initial stage From the ‡Biomedical Proteomics Research Group and §Proteome Informatics Group, Department of Structural Biology and Bioinformatics, University Medical Centre, University of Geneva, 1211 Geneva 4, Switzerland and ¶Center for Integrative Genomics, University of Lausanne, Quartier Sorge, 1015 Lausanne-Dorigny, Switzerland Received, September 21, 2009, and in revised form, October 5, 2009 Published, MCP Papers in Press, November 6, 2009, DOI 10.1074/ mcp.M900439-MCP200 1 The abbreviations used are: PTM, post-translational modification; apoA-I, apolipoprotein A-I; BAC, boronate affinity chromatography; GIL, glycation isotopic labeling; HbA1c, glycated hemoglobin; HCD, higher energy collisional dissociation; HSA, human serum albumin; TEAB, triethylammonium hydrogen carbonate buffer. Research