Pyridoxamine promotes heat-induced protein modifications in whey as evaluated by structure- and binding site-specific...

Thermal processing of whey-based foods leads to nonenzymatic post-translational protein modifications (nePTMs), which reduce the protein quality. Because pyridoxamine inhibits protein glycation and oxidation in vivo and in model reactions, the present study investigated the effect of pyridoxamine on β-lactoglobulin modifications in heated whey. After Glu-C digestion, 14 nePTMs were quantified at 29 binding sites by micro-liquid chromatography-electrospray ionization-tandem mass spectrometry in scheduled multiple reaction monitoring mode. Unexpectedly, comprehensive structure- and binding site-specific monitoring revealed that pyridoxamine promoted primary and advanced Maillard products, oxidation-, deamidation-, and deamination products in a concentration-dependent manner in whey. At the highest tested pyridoxamine concentration, 32 modifications were promoted, 6 not affected and 3 inhibited. In particular, the N-terminal modifications 4-imidazolidinone (26.7-fold change) and α-ketoamide (8.0-fold change) were most significantly enhanced by pyridoxamine; corresponding reaction mechanisms were proposed. Thus, the supplementation of whey products with pyridoxamine may even promote protein damage during heating.