Analysis of Glycated and Ascorbylated Proteins by Gas Chromatography−Mass Spectrometry

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Abstract
Proteins or poly-l-lysine which were incubated in the presence of ascorbic acid, dehydroascorbic acid (ascorbylation), or various sugars (glycation) were analyzed by gas chromatography−mass spectrometry (GC−MS). To also detect more labile reaction products, the Maillard modified proteins or poly-l-lysine were enzymatically hydrolyzed and reacted with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide to form the N(O)-tert-butyldimethylsilyl (tBDMS) derivatives prior to GC analysis. Under these conditions, the known Maillard products N ε-(carboxymethyl)lysine (1), oxalic acid mono-N ε-lysinylamide (2), and N ε-(carboxyethyl)lysine (3) could be simultaneously detected and quantified in glycated and ascorbylated proteins. Additionally, N ε-(1-carboxy-3-hydroxypropyl)-l-lysine (4) was identified for the first time as a Maillard product of proteins. Under the conditions applied here, 4 was found only in ascorbylated proteins or poly-l-lysine, but not in glycated proteins. Maillard-modified poly-l-lysine was further subjected to high-performance liquid chromatography (HPLC) analysis after enzymatic hydrolysis and formation of the phenyl isothiocyanate derivatized amino acids. Using this method, N ε-formyl-l-lysine (5), which cannot be distinguished from 2 by GC−MS analysis, was identified for the first time as a glycation product. Compound 5 is mainly formed from ribose, lactose, and fructose. The indicated Maillard products were quantified in β-lactoglobulin (GC−MS) or poly-l-lysine (HPLC) which were glycated or ascorbylated using different precursors. Keywords: Maillard reaction; glycation; ascorbylation; ascorbic acid; GC−MS

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