Oxidation of α-lactalbumin after a lactoperoxidase-catalysed reaction: An oxidomics approach applying immuno-spin trapping and mass spectrometry

Free radicals were characterised by mass spectrometry (MS) and immuno-spin trapping on α-lactalbumin (α-lac) after a lactoperoxidase (LPO)-catalysed reaction. Although spin traps have historically been used for electron spin resonance (ESR) studies, here free radicals were detected by immuno-spin trapping using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), Western blotting, and MS; the latter yields information about the location of radical formation in the protein structure. Radical formation was highly specific towards Tyr residues; DMPO-trapped radicals were detected at Tyr18 and Tyr50, and the results also indicated a radical on Tyr36. These three oxidation sites on α-lac are all exposed on the surface of the protein, indicating that accessibility for LPO or DMPO was important. Applying immuno-spin trapping in combination with MS is a good approach to understand how different oxidation mechanisms affect the generation of radicals on proteins, and this oxidomics approach has future potential in food science.