Tandem mass spectrometry for the study of glyoxal-derived advanced glycation end-products (AGEs) in peptides.

RATIONALE

The post-translational modification known as glycation affects the physiological properties of peptides and proteins. Glycation is particularly important during hyperglycaemia where α-dicarbonyl compounds are generated. These compounds react with proteins to generate α-dicarbonyl-derived glycation products, which are correlated with diabetic complications such as nephropathy, retinopathy, and neuropathy, among others. One of these α-dicarbonyl compounds is ethanedial, also known as glyoxal. Thereby, glyoxal binding to protein/peptides is studied by electron capture dissociation (ECD) and collisionally activated dissociation (CAD).

METHODS

Acetylated and non-acetylated undecapeptides containing one lysine and one arginine susceptible of glycation were reacted with glyoxal under pseudo-physiological and MeOH/H2O (50:50) conditions. Two types of glyoxal-derived AGEs were fragmented by ECD and CAD using 12 Tesla Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS).

RESULTS

Reaction with glyoxal under different reaction conditions showed the addition of C2O and C2H2O2, which corresponded to a net increase on the peptide mass of 39.9949 Da and 58.0055 Da, respectively. The binding site was assigned within an error <1 ppm, using ECD and CAD. The results indicated that both types of glyoxal-derived AGEs are formed at the side chain of arginine located in position 3.

CONCLUSIONS

Types and binding sites of glyoxal-derived AGEs were investigated in peptides containing one arginine-one lysine using FTICRMS. Two net mass additions to the mass of the peptide were assigned as C2O and C2H2O2, which were located at the arginine side chain. In addition, these mass additions (C2O and C2H2O2) observed in the peptides were unaffected by different reaction conditions.