Investigations on Protein Nonenzymatic Glycation by a New and Effective Mass Spectrometric Method

Abstract

Protein nonenzymatic glyco-oxidation has been studied eitherin vitroorin vivoby matrix assisted laser desorption/ionization (MALDI). Bovine serum albumin and ribonuclease have been incubated in pseudophysiological conditions with different sugar (glucose or fructose) concentrations. Mass increases of incubated proteins have been determined by MALDI and related to different numbers of sugar molecules condensed on the active sites of the protein. The kinetics of glyco-oxidation has been determined for the various sugar concentrations showing in all cases the reaching of a steady-state, corresponding to the complete saturation of the reactive sites. The same methodology has been applied in the study of human serum albumin from healthy subjects and well-controlled and badly controlled diabetic patients. Also in this case, clear differences have been evidenced among the three groups of subjects under investigation. In the case of badly controlled patients, the molecular weight of human serum albumin shows increases with respect to genuine human serum albumin, in the range 439–2403, corresponding to the condensation on that protein from 3 to 15 glucose units.