Analysis of the Structure and Antigenicity in Ovalbumin Modified with Six Disaccharides Through Liquid Chromatography-High-Resolution Mass Spectrometry.

Melibiose, cellobiose, maltose, lactose, turanose, and isomaltulose were selected to be glycated with OVA. The number of free amino groups of OVA modified with different disaccharides decreased, and the secondary and tertiary structures of the modified OVA also changed greatly. Moreover, the glycation sites detected by HPLC-HCD-MS/MS were all on the sensitized epitopes of OVA, which reduced the binding ability of IgG and IgE of glycated OVA. In addition, the glycation sites with the highest DSP in different samples were located in the irregular coil region of OVA. Among the six disaccharides, the glycation reaction between melibiose and OVA was the most obvious. Through the analysis of disaccharide configuration, it was found that the glycation efficiency of the reducing disaccharide linked by a 1 → 6 glycoside bond was higher than that by a 1 → 4 glycoside bond, and reducing sugar with ß type was better than that with α type. These findings would provide a theoretical reference for the use of different sugars in food production.

Investigation of the effect of oxidation on the structure of ß-lactoglobulin by high resolution mass spectrometry.

In this work, high-resolution mass spectrometry was used to identify the oxidation sites and forms of ß-lactoglobulin (ß-Lg) induced by hydrogen peroxide with 1.5% concentration, and the influence of oxidation sites on the structure of ß-Lg was discussed from the molecular level. Twelve kinds of oxidation products and 36 oxidation sites were identified, including sulfoxidation in sulfur-containing amino acid residue, hydroxylation in aromatic group residue, deamination in amino-containing amino acid etc. The destruction of hydrogen bonds and disulfide bonds in ß-Lg caused by oxidation is the main factor causing its structural changes, which were manifested in the decrease of ß-sheet component and increase of ß-turns and random coil contents, intrinsic fluorescence intensity and surface hydrophobicity. In addition, several peptides as potential oxidative markers were found to be capable of monitoring the degree of oxidation of ß-Lg. In short, this work provided insights into structural changes of ß-Lg by oxidation.

Enzymolysis Reaction Kinetics and Liquid Chromatography High-Resolution Mass Spectrometry Analysis of Ovalbumin Glycated with Microwave Radiation.

Microwave radiation was adopted to accelerate glycation between ovalbumin (OVA) and d-glucose. We evaluated the digestibility of glycated OVA from the perspective of kinetics, using pepsin and trypsin as model enzymes. Hydrolysed protein concentrations, enzymolysis kinetics, and activation energy (Ea) were investigated. The results showed that, under the conditions of simulating human digestion, the hydrolysis rate of OVA by pepsin was faster than that by trypsin, but for digestive enzymes, the digestion efficiency of OVA hydrolyzed by trypsin was higher. It was found that the rate constant of enzymatic hydrolysis of OVA was independent of the initial concentration of OVA but related to the type of protease and temperature. The reaction rate constants of glycated OVAs were significantly higher than that of native OVA during enzymolysis. Ea required for glycated OVA enzymatic hydrolysis by pepsin decreased, while that required by trypsin enzymatic hydrolysis nearly doubled. Liquid chromatography high-resolution mass spectrometry revealed that sample 1 had three glycated sites (R111, K227, and K264), sample 2 had two glycated sites (K207 and K323), sample 3 had five glycated sites (R127, R159, K227, R340, and K370), sample 4 had three glycated sites (R85, R143, and K323), and sample 5 had two glycated sites (R51 and R59). These sites increased Ea required for enzymatic hydrolysis of glycated OVA by trypsin.

Identification of glycated sites in ovalbumin under freeze-drying processing by liquid chromatography high-resolution mass spectrometry.

The glycation reaction between ovalbumin and d-glucose during freeze-drying was investigated and the mechanism of protection of the protein structure was studied, the precise glycated sites and degree of substitution per peptide (DSP) of each site were determined using liquid chromatography high-resolution mass spectrometry. It was found that lysine residues are the main glycated sites under freeze-drying. K62 and K264 were the most reactive glycated sites in lyophilized ovalbumin, with a DSP close to 80%. The glycated sites were located at the outer surface of the global protein. The unglycated sites were located at the outer surface of the hydrophobic pocket and in the six main strands of the ß-sheet. Therefore, the glycation reaction of the protein was occurred in the solvent accessible area. It was hypothesized that few changes occurred in the conformation to disturb the glycated sites under freeze-drying. In particular, the main strands of the ß-sheet of ovalbumin were more stable. Freeze-drying was a mild process and protected the conformation without extensive denaturation.