MassIVE MSV000088240

Partial Public PXD029170

Site-specific characterization of heat-induced disulfide rearrangement in beta-lactoglobulin by liquid chromatography-mass spectrometry

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Description

Disulfides are important for maintaining protein native structure, but they may undergo rearrangement in the presence of free Cys residues, especially under elevated temperatures. Disulfide rearrangement may result in protein aggregation, which is associated with in vivo pathologies in organisms as well as in vitro protein functionality in food systems, e.g. during thermal processing. In the present study, an LC-MS-based bottom-up site-specific proteomic approach was optimized to study disulfide rearrangements in beta-lactoglobulin, b-LG, under different heat treatments (60-90 C). Artifactual disulfide rearrangement observed during sample preparation using a conventional protocol was minimized by blocking the remaining free Cys residues after heat treatment of the samples. Use of Endoproteinase Glu-C for enzymatic hydrolysis allowed the identification and comparison of relative intensity of all theoretically possible disulfide cross-links formed by the heat treatments. Non-native disulfides were formed from heat treatment at approx. 70 C where b-LG started to unfold, while higher levels of inter-molecular disulfide links were formed at and higher than 80 C, in agreement with b-LG aggregation detected by SEC analysis. Collectively, the abundant signal of Cys66-containing non-native disulfide links after heating suggested that Cys66 is a key disulfide-linked Cys residue in b-LG participating in heat-induced inter-molecular disulfide links, and the corresponding protein aggregation. [doi:10.25345/C5TZ8N] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: beta-lactoglobulin ; alpha-lactalbumin

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Principal Investigators:
(in alphabetical order) 		Marianne Nissen Lund, Department of Food Science, University of Copenhagen, Denmark
Submitting User: chengkang
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