Identification of lanthionine and lysinoalanine in heat-treated wheat gliadin and bovine serum albumin using tandem mass spectrometry with higher-energy collisional dissociation.

The present manuscript reports on the identification of various dehydroamino acid-derived bonds and cross-links resulting from thermal treatment (excess water, 240 min, 130 °C) of two model food proteins, bovine serum albumin, and wheat gliadin. S-Carbamidomethylated tryptic and chymotryptic digests of unheated (control) and heated serum albumin and gliadin, respectively, were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-ESI-MS/MS) with higher-energy collisional dissociation (HCD). Heat-induced ß-elimination of cystine, serine and threonine, and subsequent Michael addition of cysteine and lysine to dehydroalanine and 3-methyl-dehydroalanine were demonstrated. Lanthionine, lysinoalanine, 3-methyl-lanthionine, and 3-methyl-lysinoalanine were identified. The detection of inter-chain lanthionine in both bovine serum albumin and wheat gliadin suggests the significance of these cross-links for food texture.

N-ε-fructosyllysine and N-ε-carboxymethyllysine, but not lysinoalanine, are available for absorption after simulated gastrointestinal digestion.

Food processing leads to a variety of chemical modifications of amino acids in food proteins. Recent studies have shown that some modified amino acids resulting from glycation reactions can pass the intestinal barrier when they are bound in dipeptides. In this study, we investigated as to what extent modified amino acids are released from post-translationally modified casein during simulated gastrointestinal digestion. Casein was enriched with N-ε-fructoselysine, N-ε-carboxymethyllysine, and lysinoalanine, in different degrees of modification. The casein samples were subjected to a two-step proteolysis procedure, simulating gastrointestinal digestion. The digestibility of modified casein as measured by analytical size-exclusion chromatography (SEC) decreased with increasing degree of modification especially after enrichment of fructoselysine and lysinoalanine. Semi-preparative SEC of digested casein samples revealed that fructoselysine and carboxymethyllysine are released bound in peptides smaller than 1,000 Da, which is comparable to native amino acids. The glycation compounds should, therefore, be available for absorption. Lysinoalanine as a crosslinking amino acid, however, is mostly released into longer peptides of at least 30-40 amino acids which should strongly impair its absorption availability.

Dehydroalanine crosslinks in human lens.

This study was conducted to develop a methodology for the purification and detection of histidinoalanine, lanthionine and lysinoalanine in the lens tissue. Cataractous and aged human lens proteins were hydrolysed and fractionated by using anion-exchange chromatography. The fraction containing the bulk of dehydroalanine crosslinks was derivatized with dansyl chloride and then separated and quantified by means of RP-HPLC. The spectral and chromatographic properties of all three substances purified and quantified in this study were identical to those of their synthesized counterparts. Histidinoalanine and lanthionine were the most abundant dehydroalanine crosslinks in both water-soluble and water-insoluble lens proteins. Histidinoalanine levels in water-soluble proteins from the cataractous lenses of Indian origin were 6.2-fold higher than those in water-soluble proteins from normal lenses (1.68+/-0.75 vs 0.26+/-0.06 nmol/mg protein; p<0.001). In water-insoluble proteins, they were 2.2-fold higher in cataractous lenses compared with normal lenses (1.59+/-0.76 vs 0.73+/-0.17 nmol/mg protein; p<0.01). Lanthionine levels were significantly higher in water-insoluble proteins of cataractous lenses when compared to non-cataractous lenses (2.5+/-1.68 vs 0.95+/-0.08 nmol/mg protein; p<0.03). Unlike histidinoalanine, this crosslink appears to accumulate in relatively high concentrations in water-soluble lens proteins; its concentration was 9-fold higher than histidinoalanine from the same proteins (0.26+/-0.06 HAL vs 2.34+/-0.76 LAN nmol/mg protein; p<0.0004). The concentration of lysinoalanine was in the picomolar range and in cataractous lens proteins only.