Analysis of glycated and ascorbylated proteins by gas chromatography-mass spectrometry.
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 (epsilon)-(carboxymethyl)lysine (1), oxalic acid mono-N (epsilon)-lysinylamide (2), and N (epsilon)-(carboxyethyl)lysine (3) could be simultaneously detected and quantified in glycated and ascorbylated proteins. Additionally, N (epsilon)-(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 (epsilon)-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 beta-lactoglobulin (GC-MS) or poly-L-lysine (HPLC) which were glycated or ascorbylated using different precursors.
Hasenkopf
Katrin
K
Institute of Pharmacy and Food Chemistry, Friedrich-Alexander-University, Erlangen-Nürnberg, Schuhstrasse 19, Germany.
Rönner
Birgit
B
Hiller
Hartmut
H
Pischetsrieder
Monika
M
D001205:Ascorbic Acid / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D003683:Dehydroascorbic Acid / Q000737:chemistry*; D005632:Fructose / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry; D006023:Glycoproteins / Q000032:analysis*; D006868:Hydrolysis; D007782:Lactoglobulins / Q000032:analysis; D007785:Lactose / Q000737:chemistry; D015416:Maillard Reaction; D013058:Mass Spectrometry; D011107:Polylysine / Q000032:analysis / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D012266:Ribose / Q000737:chemistry
D006023:Glycoproteins; D007782:Lactoglobulins; D011506:Proteins; D011107:Polylysine; D005632:Fructose; D012266:Ribose; D007785:Lactose; D001205:Ascorbic Acid; D003683:Dehydroascorbic Acid