Ultra performance liquid chromatography-mass spectrometric determination of the site specificity of modification of b-casein by glucose and methylglyoxal
Lima, M., Moloney, C. and Ames, J. (2009) Ultra performance liquid chromatography-mass spectrometric determination of the site specificity of modification of b-casein by glucose and methylglyoxal. Amino Acids, 36 (3). pp. 475-481. ISSN 0939-4451 Available from: http://eprints.uwe.ac.uk/17114
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Publisher's URL: http://dx.doi.org/10.1007/s00726-008-0105-y
Modification of protein by carbonyl compounds under in vitro physiological conditions is sitedirected. There are few reports of the site specificity of glycation of proteins using heating conditions of relevance to food processing. The aim of this study was to determine the site specificity of modification of b-casein (bCN) by glucose and methylglyoxal (MGO). bCN (1.33 M, 3.2%) was heated with either glucose (1.345 M, 4.6%) or MGO (1 mM) at 95C for up to 4 h. Tryptic digests were prepared and analysed by ultra performance liquid chromatography electrospray ionisation mass spectrometry (UPLC-ES/MS). The sites of formation of the Amadori product, Ne-(fructosyl)lysine (FL), and the advanced glycation end-products, Ne-(carboxymethyl)lysine (CML), MGO-derived dihydroxyimidazolidine (MG-DH) and MGO-derived hydroimidazolone (MG-HI), were located. FL and CML were detected at K107 and K176 residues in bCN/glucose incubations. Indigenous Ne-(lactulosyl)lysine was detected at K107 only. MG-DH and MG-HI were detected at R202 and possibly R183 residues in both bCN/ glucose and bCN/MGO incubations. Glycation of bCN by glucose and MGO resulted in similar site specificity for MG-DH and MG-HI formation.