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

• Using sodium borohydride or sodium borodeuteride to introduce stable isotopes to peptides containing glycation.
• The introduced isotopes were used for identification and relative quantitation.
• A good correlation between glycation and solvent exposure was established for human IgG1-Fc.
• This method can be applied where using stable isotope labeled glucose cannot be applied.

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.