Oxidation of Whey Proteins during Thermal Treatment Characterized by a Site-Specific LC–MS/MS-Based Proteomic Approach

Abstract

Thermal treatment is often employed in food processing to tailor product properties by manipulating the ingredient functionality, but these elevated temperatures may accelerate oxidation and nutrient loss. Here, oxidation of different whey protein systems [α-lactalbumin (α-LA), β-lactoglobulin (β-LG), a mix of α-LA and β-LG (whey model), and a commercial whey protein isolate (WPI)] was investigated during heat treatment at 60−90 °C and a UHT-like treatment by LC-MS-based proteomic analysis. The relative modification levels of each oxidation site were calculated and compared among different heat treatments and sample systems. Oxidation increased significantly in protein systems after heating at ≥90 °C but decreased in systems with higher complexity [pure protein (α-LA > β-LG) > whey model > WPI]. In α-LA, Cys, Met, and Trp residues were found to be most prone to oxidation. In β-LG-containing protein systems, Cys residues were suggested to scavenge most of the reactive oxidants and undergo an oxidation-mediated disulfide rearrangement. The rearranged disulfide bonds contributed to protein aggregation, which was suggested to provide physical protection against oxidation. Overall, limited loss of amino acid residues was detected after acidic hydrolysis followed by UHPLC analysis, which showed only a minor effect of heat treatment on protein oxidation in these protein systems.

Cysteine residues are responsible for the sulfurous off-flavor formed in heated whey protein solutions

“…It was hypothesized that these long peptides, having multiple reactive amino acids, may have a high possibility to undergo different modifications (e.g. oxidation) simultaneously under elevated temperatures, leading to a lower probability of having only Cys modified to DHA, especially in α-LA ( Li, Nielsen, Engholm-Keller, & Lund, 2022 ). In addition, the tryptic β-LG tri-Cys peptide [102–124] from mixed protein systems under the non-reduced condition was likely to be involved in disulfide-linked peptides, resulting in the absence of signal of the targeted linear DHA-modified peptide in the present proteomic analysis.…”

“…All protein samples were also prepared in 55 mM HEPES buffer (pH 7.0) for proteomic and thiol analyses. Protein and amino acid concentrations in the prepared solutions were chosen to reflect their concentrations in a 3 % WPI (w/w) solution ( Li, Nielsen, Engholm-Keller, & Lund, 2022 ).…”

“…Peptide modifications of samples from the non-reduced group were identified by the Proteome Discoverer software (ver. 2.2.0.388, Thermo Fisher Scientific Inc., Roskilde, Denmark) with previously described settings ( Li, Nielsen, Engholm-Keller, & Lund, 2022 ). Cys → DHA (DHA) (-34 Da), Cys → persulfide carbamidomethylation (persulfide) (+89 Da) and Cys carbamidomethylation (alkylation) (+57 Da) were chosen as variable modifications, while N -term → pyruvic acid (–33 Da) and C-term → amidation (-104 Da) were selected as variable modifications at any peptide N -terminus and C-terminus, respectively ( Supporting Information , Figure S1 ).…”

“…Database search results (.msf file) were validated in the Skyline software (ver. 20.1.0.76, University of Washington, Seattle, WA, USA) by manually checking retention time, isotopic correlation and fragment ion distribution for each modification ( Li, Nielsen, Engholm-Keller, & Lund, 2022 , Schilling et al, 2012 ). Databases containing β-LG (P02754) or α-LA (P00711) (downloaded from Uniprot [ http://www.uniprot.org ], accessed in September 2019) were selected for samples of β-LG and α-LA, respectively, while their combination was used for searches of the whey model and WPI.…”

“…Statistical analysis was performed by one-way ANOVA with a Tukey-Kramer HSD post-hoc test in JMP Pro 15 (SAS Institute Inc., Cary, North Carolina, USA). All MS.raw data in the present study was the same as in our previously published papers ( Li et al, 2022 , Li, Nielsen, Engholm-Keller, & Lund, 2022 ), where the extent of disulfide rearrangement and protein oxidation was investigated. The modification search results are available via MassIVE ( https://massive.ucsd.edu ) with identifier MSV000088811.…”