Priego-Capote, Feliciano

Ramírez-Boo, M.

Scherl, Alexander

Sánchez, Jean-Charles

Repositorio Institucional de la Universidad de Córdoba

72 3527(Ï0,&$1Ò0(52)(%5(52$PRQJSRVWWUDQVODWLRQDOPRGL¿FDWLRQV370Vof proteins, non-enzymatic glycation is one of the less frequently studied in proteomics. Glycated pro-teins are formed by a non-enzymatic reaction bet-ween reducing carbohydrates (e.g., glucose, fructo-se, ribose or derivatives such as ascorbic acid) with amino groups located in N-terminal position or in ly-sine and arginine residues (Figure 1) [1]. Analytical guidelines required to succeed in the characterization of glycated proteins at qualitative levels have been UHFHQWO\SURSRVHG>@,WLQYROYHVWKHLGHQWL¿FDWLRQof glycated proteins as well as the elucidation of su-gar attachment sites. Nevertheless, there is a demand for quantitative proteomic strategies that could be potentially applied for glycation assessment. One of WKHDUHDVWKDWFRXOGEHQH¿WIURPWKHVHVWUDWHJLHVLVWKHFOLQLFDO¿HOGGXHWRWKHFUXFLDOUROHRIJOXFRVHDVan energy source in humans, being the main circu-lating sugar and, thus, the most relevant molecule in terms of protein glycation. In fact, there are clinical evidences relating glycation and pathological condi-tions associated to hyperglycaemia such as diabetes mellitus, renal failure or aging [5-7].Strategies for proteomic analysis of blood glycated proteinsMaría Ramírez-Boo1, Feliciano Priego-Capote2, Alexander Scherl1, Jean-Charles Sanchez11 Biomedical Proteomics Research Group, Dpt. of Structural Biology and Bioinformatics, University Medi-cal Center, Geneva, Switzerland. 2 Dpto. de Química Analítica, Universidad de Córdoba, Spain.A set of innovative approaches for analysis of glycated proteins is here presented by application to blood samples [8]. Qualitative analysis was carried out by tandem mass spectrometry (MS) after en-GRSURWHLQDVH*OX&GLJHVWLRQDQGERURQDWHDI¿QLW\chromatography (BAC) for isolation of glycated pep-tides. For this purpose, two MS operational modes were used: HCD-MS2 and CID-MS3 by neutral loss scan (monitoring two selective neutral losses typical RISHSWLGHVPRGL¿HGE\JOXFRVHDWWDFKPHQWand 84.04 Da). Quantitative analysis was based on the labelling of proteins with 13C6-glucose incubation in order to evaluate the native glycated proteins labelled with 12C6-glucose. As glycation is chemo-selective, it is exclusively occurring in potential targets for in vivo PRGL¿FDWLRQV7KLVDSSURDFKQDPHG*O\FDWLRQ,VRWR-pic Labelling (GIL), enabled to differentiate glycated peptides labelled with both isotopic forms resulting from enzymatic digestion by MS (6 Da mass shift per glycation site) as shown in Figure 2.:LWK WZRGLIIHUHQW SXUSRVHV WKLV VWUDWHJ\KDVbeen applied to human plasma and lysates from Figure 1. Scheme of the glycation process (Maillard reaction).3527(Ï0,&$1Ò0(52)(%5(52 73Figure 2. %RWWRPXSSURWHRPLFVZRUNÀRZVXVHGIRUTXDQWLWDWLYHDQDO\VLVRIJO\FDWHGSURWHLQVVKRZLQJWKHODEHOOLQJZLWKlight and heavy glucose.74 3527(Ï0,&$1Ò0(52)(%5(52HU\WKURF\WHV7KH¿UVWRQHZDVEDVHGRQWKHFKDUDF-terization of the native glycated proteome, incuba-ting the clinical samples with 13C6-glucose prior to DQDO\VLVZLWKWKHFRQYHQWLRQDOSURWHRPLFVZRUNÀRZexposed above. Fifty glycated proteins with identi-¿FDWLRQRIVXJDUDWWDFKPHQWSRVLWLRQVZHUHGH-tected in the analysis of human plasma. Concerning UHGEORRGFHOOVJO\FDWHGSURWHLQVZLWKLGHQWL¿FD-tion of 54 glycation sites were detected without any protein pre-fractionation step. These proteins are representative targets to compare between samples with different glycaemic states and monitor the gly-caemic control. The second task was focused on the assessment of glucose stimuli at different concen-trations (0, 5, 10, 30 and 50 mM glucose) in human SODVPD)RUWKLVSXUSRVH¿YHSODVPDDOLTXRWVZHUHindependently incubated with 0, 5, 10, 30 and 50mM 12C6-glucose for 24 h and, subsequently mixed with a plasma aliquot incubated with 30mM 13C6-glucose for 24 h. Proteomic analysis of these pools has re-vealed additional information about the biological effect of different hyperglycemia conditions and the kinetic of glycation.References[1] Maillard, L. C. Action des acides amines sur le sucres: formation des mélanoidines per voie mé-thodique. C. R. Acad. Sci. 1912; 154, 66-68.[2] Zhang, Q., Tang, N., Schepmoes, A. A., Phillips, L. S., Smith, R. D., Metz, T. O. Proteomic pro-¿OLQJRIQRQHQ]\PDWLFDOO\JO\FDWHGSURWHLQVLQhuman plasma and erythrocyte membranes. J. Proteome Res. 2008; 7, 2025-2032.[3] Zhang, Q., Petyuk, V. A., Schepmoes, A. A., Orton, D. J., Monroe, M. E., Feng, Y., Smith, R. D., Metz, T. O. Analysis of non-enzymatically glycated peptides: neutral-loss-triggered MS3 versus multi-stage activation of tandem mass spectrometry. Rap. Comm. Mass Spectrom. 2008; 22, 3027-3034.[4] Zhang, Q., Ames, J. M., Smith, R. D., Baynes, -:0HW]72$SHUVSHFWLYHRQWKH0DLOODUGreaction and the analysis of protein glycation by mass spectrometry: probing the pathogenesis of chronic disease. J. Proteome Res. 2009; 8, 754-769.>@ %D\QHV-:7KHUROHRI$*(VLQDJLQJFDX-sation or correlation. Exp. Gerontol. 2001; 36, 1527-1537.[6] Brownlee, M. Biochemistry and molecular cell biology of diabetic complications. Nature 2001; 14, 813-820.[7] Hipkiss, A. R. Accumulation of altered proteins and ageing: causes and effects. Exper. Gerontol. 2006; 41, 464-473.[8] Priego-Capote F., Scherl$0OOHU0:DULGHOP., LisacekF., Jean-Charles SanchezJ-C. Glyca-tion Isotopic Labelling (GIL) with 13C-reducing Sugars for Quantitative Analysis of Glycated Pro-teins in Human Plasma MCP, 2009, in press.

Strategies for proteomic analysis of blood glycated proteins

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