Site-Specific Characterization of Heat-Induced Disulfide Rearrangement in Beta-Lactoglobulin by Liquid Chromatography–Mass Spectrometry
Chengkang Li, Kasper Engholm‐Keller, Marianne N. Lund
Abstract
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 (β-LG) under different heat treatments (60-90 °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 β-LG disulfide cross-links formed by the heat treatments. Non-native disulfides were formed from heat treatment at approx. 70 °C where β-LG started to unfold, while higher levels of inter-molecular disulfide links were formed at ≥80 °C, in agreement with β-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 β-LG participating in heat-induced inter-molecular disulfide bonds and the corresponding protein aggregation.