Identification and comparative quantitation of glycation by stable isotope labeling and LC–MS

Glycation is a common modification of proteins both in vitro and in vivo. To aid identification and comparative quantitation, a method of stable isotope labeling followed by LC–MS analysis was proposed. The samples were reduced using sodium borohydride or sodium borodeuteride. Reduction of the Schiff base between the amine group and the reducing sugars resulted in a molecular weight increase of 2 Da using sodium borohydride or a molecular weight increase of 3 Da using sodium borodeuteride. The molecular weight difference of 1 Da between peptides containing glycated lysine residue reduced using sodium borohydride or sodium borodeuteride was used to identify glycated peptides and to calculate the glycation difference between samples. The method was used to investigate glycation of a recombinant human IgG1 antibody under native and denaturing conditions. The result demonstrated a good correlation between glycation propensity of lysine residues and their solvent exposure levels.