Monitoring protein glycation by electrospray ionization (ESI) quadrupole time-of-flight (Q-TOF) mass spectrometer.

In this study electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) mass spectrometry was used to investigate protein glycation. The glycated species of cytochrome C, lysozyme, and ß-casein formed during glycation with d-glucose were identified and monitored in binary systems heated at 70°C under dry and aqueous conditions. Cytochrome C had multiple charges in non-glycated state, primarily changing from +13 to +17 positive charges, whereas ß-casein had charge states up to +30. Upon heating with glucose at 70°C in aqueous state, attachment of one glucose molecule onto proteins was observed in each charge state. However, heating in dry state caused much more glucose attachment, leading to the formation of multiple glycoforms of proteins. By using ESI-QTOF-MS technique, formation of glycated cytochrome C containing up to 12 glucose moieties were observed, while glycated species containing 6 and 8 glucose moieties were observed for lysozyme and ß-casein, respectively in various heating conditions.

Kinetic evaluation of the inhibition of protein glycation during heating.

This study aimed to investigate the kinetics of early stage of the Maillard reaction by a reversible bimolecular reaction mechanism and also to evaluate the compatibility of enzyme inhibition kinetics for calculating the inhibitory activity of protein anti-glycation agents. Model systems composed of ovalbumin, glucose, and anti-glycation agents (tannic acid or calcium ion) at different molar ratios were heated at 90 °C for different times in dry state or in solution. Heated samples were analysed for furosine, acid derivative of N-ε-fructoselysine (FL), to monitor the progression of the early glycation stage. Compared to a control, presence of calcium ions and tannic acid decreased FL formation significantly (p<0.05) during heating in dry state. Evaluation of the kinetic data revealed that calcium inhibited glycation of ovalbumin by a mixed non-competitive mechanism in both dry and in solution conditions; while the mode of inhibition by tannic acid was found to be purely non-competitive in the dry state.

Effects of hydrophobic and ionic interactions on glycation of casein during Maillard reaction.

This study aimed to investigate the effects of hydrophobic and ionic interactions on glycation of native and high-shear treated casein during heating. Casein-epicatechin and casein-calcium complexes were formed and glycated with glucose at different temperatures ranging from 70 to 150 °C in solution and dry states. Furosine, acid derivative of N-ε-fructoselysine (FL), and N-ε-carboxymethyl lysine (CML) were measured as indicators of early and advanced glycation, respectively. CML concentrations of casein-epicatechin and casein-calcium complexes heated in solution were significantly lower as compared to the control (p < 0.05). For instance, 182 ± 9.78 µg/g of CML formed in the control, while CML concentrations were 136 ± 10.7 and 101 ± 7.37 µg/g in casein-epicatechin and casein-calcium complexes, respectively, heated at 150 °C in the solution state. Treatment by high shear microfluidization further decreased the CML formed during heating at 70 °C in dry state. The results suggest that interactions with epicatechin molecule and calcium ion could be a useful strategy to limit advanced glycation of casein under certain conditions.