A liquid chromatography–mass spectrometry method based on multiple reaction monitoring (MRM) was developed for the simultaneous quantification of markers representing two potentially competing pathways, the Maillard reaction and the dehydroalanine pathway. The two pathways involve the same residues in the proteins to some extent, namely, the essential amino acid lysine, as well as free-amino terminals available on proteins and polypeptides, competition between the two pathways in food systems may occur. The developed method comprises the following markers of the Maillard reaction: furosine, N-ε-(carboxyethyl)lysine (CEL) and N-ε-(carboxymethyl)lysine (CML), together with the dehydroalanine reaction pathway markers; lanthionine (LAN) and lysinoalanine (LAL), as well as lysine itself. The validated method was then used for the absolute quantification of heat-induced protein modifications in model systems of micellar casein and whey protein isolates (MCI and WPI, respectively) in the presence or absence of lactose. As expected, the Maillard reaction markers furosine, CEL and CML increased during the applied heat treatment in the presence of lactose, whereas the dehydroalanine markers, LAN and LAL increased with heating in both MCI and WPI, both in the presence and absence of lactose, although at lower levels in the presence of lactose, confirming the competing state of the two pathways.
- advanced glycation end products
- multiple reaction monitoring
- protein modification