Site-Specific Characterization of Heat-Induced Disulfide Rearrangement in Beta-Lactoglobulin by Liquid Chromatography-Mass Spectrometry

Disulfides are important for maintaining the 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 and in vitro protein functionality in food systems. In a food context, it is therefore important to understand the process of disulfide rearrangement on a site-specific level in order to control aggregation. In the present study, a liquid chromatography-mass spectrometry (LC-MS)-based bottom-up site-specific proteomic approach was optimized to study disulfide rearrangements in beta-lactoglobulin (beta-LG) under different heat treatments (60-90 degrees C). Artifactual disulfide rearrangement observed during sample preparation using a conventional protocol was detected and minimized by blocking the remaining free Cys residues with iodoacetamide in the presence of urea after heat treatment. Use of endoproteinase Glu-C for enzymatic hydrolysis allowed, for the first time, identification and comparison of the relative intensity of all theoretically possible beta-LG disulfide cross-links formed by the heat treatments. Non-native disulfides were formed from heat treatment at approx. 70 degrees C where beta-LG started to unfold, while higher levels of inter-molecular disulfide links were formed at >= 80 degrees C, in agreement with beta-LG aggregation detected by size exclusion chromatography analysis. Collectively, the Cys residues of the surface-located native disulfide Cys66-Cys160 were proposed to be more reactive, participating in heat-induced disulfide rearrangement, compared to other Cys residues. The abundant signal of non-native disulfide bonds containing Cys66, especially Cys66-Cys66, observed after heating suggested that Cys66 is a key disulfide-linked Cys residue in beta-LG participating in heat-induced inter-molecular disulfide bonds and the corresponding protein aggregation.

Automated summary

This study optimized a LC-MS-based proteomic approach to investigate disulfide rearrangement in beta-lactoglobulin under different heat treatments. The results showed non-native disulfide bonds forming at around 70°C and increased inter-molecular disulfide links at temperatures >= 80°C, correlating with protein aggregation as detected by size exclusion chromatography. Cys66 was identified as a key residue in heat-induced disulfide rearrangement and protein aggregation in beta-LG.