Characterization of protein-polyphenol adduct formation in skim milk after addition of 4-methylcatechol

4-Methylcatechol (4MC) is an antioxidative polyphenol found in roasted coffee and cocoa powder. Here we characterize the covalent protein-polyphenol adducts formed when 4MC is added to skim milk, as determined by amino acid site-specific LC-MS/MS analysis after enzymatic digestion of milk proteins and by detection of Cys-4MC adducts after acidic hydrolysis. Three treatments of control milk samples (without addition of 4MC) and milk samples with 0.275% (w/v) 4MC added were included: (i) unheated (1 h) (ii) heated for 4 s at 140 °C (to mimic UHT treatment) (iii) heated for 300 s at 140 °C (representing an extreme heat treatment). The yield of the Cys-4MC adduct increased with increasing heating time. Interestingly, the Cys-4MC adduct was also detected in unheated milk samples with 4MC added, while the longest heating time (300 s) resulted in a nine-fold higher concentration of Cys-4MC adduct than in unheated samples. Site-specific LC-MS/MS analysis revealed 4MC-adducts on Cys, Lys, and His residues of milk proteins, with Cys being the primary target of 4MC modification (with estimated modification levels of 10.1–100%) followed by 4MC-His (0.0–0.3% modification), and 4MC-Lys (0.0–0.1% modification).