Abstract
| - The nonenzymatic glycation of proteins by reducing sugars, also known as the Maillard reaction, hasreceived increasing recognition from nutritional science and medical research. In this study, we appliedmatrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) toperform relative and simultaneous quantification of the Amadori product, which is an early glycationproduct, and of Nε-(carboxymethyl)lysine and imidazolone A, two important advanced glycation endproducts. Therefore, native lysozyme was incubated with d-glucose for increasing periods of time (1,4, 8, and 16 weeks) in phosphate-buffered saline pH 7.8 at 50 °C. After enzymatic digestion withendoproteinase Glu-C, the N-terminal peptide fragment (m/z 838; amino acid sequence KVFGRCE)and the C-terminal peptide fragment (m/z 1202; amino acid sequence VQAWIRGCRL) were usedfor relative quantification of the three Maillard products. Amadori product, Nε-(carboxymethyl)lysine,and imidazolone A were the main glycation products formed under these conditions. Their formationwas dependent on glucose concentration and reaction time. The kinetics were similar to those obtainedby competitive ELISA, an established method for quantification of Nε-(carboxymethyl)lysine andimidazolone A. Inhibition experiments showed that coincubation with Nα-acetylargine suppressedformation of imidazolone A but not of the Amadori product or Nε-(carboxymethyl)lysine. The presenceof Nα-acetyllysine resulted in the inhibition of lysine modifications but in higher concentrations ofimidazolone A. o-Phenylenediamine decreased the yield of the Amadori product and completelyinhibited the formation of Nε-(carboxymethyl)lysine and imidazolone A. MALDI-TOF-MS proved tobe a new analytical tool for the simultaneous, relative quantification of specific products of the Maillardreaction. For the first time, kinetic data of defined products on specific sites of glycated protein couldbe measured. This characterizes MALDI-TOF-MS as a valuable method for monitoring the Maillardreaction in the course of food processing. Keywords: Maillard reaction; MALDI-TOF-MS; protein glycation; Nε-(carboxymethyl)lysine; imidazoloneA; Amadori product
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