A novel LC-MS application to investigate oxidation of peptides isolated from β-lactoglobulin

Oxidation of β-lactoglobulin (β-Lg), a typical milk whey protein, was investigated by oxidizing its three tryptic peptides after separation and fractionation by preparative HPLC. Oxidation was performed with H2O2 and Fe(3+) in piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) buffer containing ascorbic acid, by keeping the samples in an oven of +37 °C for 14 days. Changes in the oxidized peptides were then analyzed with LC-ESI-QIT-MS. The peptides chosen were Ala-Leu-Pro-Met-His-Ile-Arg (ALPMHIR), Leu-Ile-Val-Thr-Gln-Thr-Met-Lys (LIVTQTMK) and Val-Leu-Val-Leu-Asp-Thr-Asp-Tyr-Lys (VLVLDTDYK), all containing amino acids of oxidative interest. Especially methionine (M) was prone to oxidize as well as dioxidize, along with tyrosine (Y), histidine (H) and/or proline (P). The ions m/z 854 [ALPMHIR + O], m/z 950 [LIVTQTMK + O] and m/z 966 [LIVTQTMK + 2O] are considered very promising indicators of β-Lg oxidation. Consequently, this study proposes a novel approach in peptide oxidation research through monitoring the oxidation markers identified with the LC-MS(n).