The Reduction in the IgE-Binding Ability of beta-Lactoglobulin by Dynamic High-Pressure Microfluidization Coupled with Glycation Treatment Revealed by High-Resolution Mass Spectrometry

摘要

Our previous study indicated that pretreatment by dynamic high-pressure microfluidization (DHPM) and glycation with galactose was a promising method for decreasing the immunoglobulin E (IgE)-binding ability of beta-lactoglobulin (beta-LG). In this work, the conformational alteration of beta-LG subjected to DHPM and glycation treatment was investigated in relation to IgE-binding ability by orbitrap mass spectrometry. After DHPM pretreatment, lower IgE-binding ability of glycated beta-LG-was observed with increasing pressures. Prior to DHPM pretreatment, 11 glycated sites were identified, while the number of glycation sites was increased to 12 after pretreatment. However, there was no significant difference of the glycation sites, at the pressures of 50, 100, and 200 MPa, respectively. Average degree of substitution per peptide molecule of beta-LG (DSP) was investigated to assess the degree of glycation per glycation site. All of the samples pretreated by DHPM exhibited a higher glycation level than those without DHPM pretreatment. The shielding effects of epitopes owing to glycation contributed to the reduction of IgE-binding capacity. Orbitrap mass spectrometry could provide a comprehensive understanding of the nature of protein glycation.