Qiuting Zhang

Glycation promoted by dynamic high pressure microfluidisation pretreatment revealed by high resolution mass spectrometry.

The effect of dynamic high pressure microfluidisation (DHPM) pretreatment on the glycation of bovine serum albumin (BSA) was investigated. A detailed glycation map was obtained from high resolution mass spectrometry. Without DHPM pretreatment, only 7 glycation sites were identified, whereas the numbers were increased to 10, 11 and 11 when BSA-glucose was pretreated with DHPM at 50, 100 and 200 MPa, respectively, suggesting that DHPM pretreatment can significantly promote the Maillard reaction. Average degree of substitution per peptide molecule BSA (DSP) was used to further evaluate the glycation level under various DHPM conditions. All the DHPM pretreated samples exhibited elevated glycation level compared to the un-pretreated sample. With 100 MPa DHPM pretreatment, the protein showed the most significantly enhanced glycation extent. In addition, our results suggest that Maillard-type glycation followed by mass spectrometry analysis can be used to study the conformational changes when proteins are disturbed by external forces.

Improved Glycation after Ultrasonic Pretreatment Revealed by High-Performance Liquid Chromatography–Linear Ion Trap/Orbitrap High-Resolution Mass Spectrometry

The glycation extent of bovine serum albumin (BSA) before and after ultrasonication was evaluated by MALDI-TOF and Orbitrap mass spectrometry. Ultrasonic pretreatment significantly improved the incorporation of galactose to BSA. Prior to ultrasonic pretreatment, only 12 sites (11 lysines and 1 arginine) were glycated, whereas the number of glycation sites was increased to 42, including 39 lysines and 3 arginines, after treatment. Average degree of substitution per peptide molecule of BSA (DSP) was used to evaluate the glycation level for each glycation site. The ultrasonic pretreatment significantly improved the DSP value of all glycation sites. The prevalently promoted glycation by ultrasonic pretreatment suggests that ultrasonication improves glycation through altering the structure of BSA throughout all three domains. An ultrahigh-resolution linear ion trap Orbitrap mass spectrometer facilitates unambiguous localization of glycation sites, allowing an in-depth analysis of the nature and extent of protein glycation at the molecular level. High-intensity ultrasonication can greatly improve protein glycation and, therefore, opens new routes to modify the functionality of proteins in a positive way.

Dynamic high pressure microfluidization-assisted extraction and antioxidant activities of lentinan.

Dynamic high pressure microfluidization (DHPM) was applied to assist the lentinan extraction. Response surface methodology (RSM), based on Box-Behnken design, was employed to optimize the DHPM-assisted extraction conditions of lentinan. Three main independent variables (DHPM pressure, ratio of water to raw material, extraction temperature) were taken into consideration. A yield of 7.200% was obtained under a modified condition (ratio of water to raw material of 65 mL/g, DHPM pressure of 147 MPa, extraction temperature of 83 °C), which matched well with the predicted value of the model. The molecular weight of the DHPM-assisted extract and hot water extract was 913,329 and 965,361 Da, respectively. Compared to the traditional hot water extraction, the lentinan extracted by DHPM assisting had better scavenging capacity of hydroxyl radical, superoxide anion free radical, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and nitrite. It could be concluded that the DHPM was a promising method to enhance the yield and antioxidant activity of lentinan during extraction.

Increase of Ovalbumin Glycation by the Maillard Reaction after Disruption of the Disulfide Bridge Evaluated by Liquid Chromatography and High Resolution Mass Spectrometry

The number of glycation sites of ovalbumin was monitored by Fourier transform ion cyclotron mass spectrometry (FTICR-MS) before and after reducing the pair of the intrachain disulfide bond. Reducing the disulfide bond of the protein greatly improved the reactivity of glycation both in dry-state and solution. The glycation sites identified by MS/MS showed that the major glycation sites of the ovalbumin were lysines. Our results suggest that glycation is strongly dependent on the protein tertiary structure, with significantly stronger reaction when the protein tertiary structure is disrupted after reducing the disulfide bond. The number of glycated sites of the protein was increased from seven to twelve in dry-state and one to two in aqueous solution. The glycation sites were found to be regulated by protein tertiary structure, hydrogen bonding, and neighboring amino acid compositions.

Probing the conformational changes of ovalbumin after glycation using HDX-MS

The conformational changes of the glycated ovalbumin were studied by hydrogen/deuterium exchange coupled with high resolution mass spectrometry technique (HDX-MS). After incubation with glucose at 50 °C for 6 h, 9 glycated peptides were detected and the corresponding glycation sites were identified. The glycation extent of each peptide was relatively high, almost over 0.5 in all peptides. A detailed peptide mapping revealed that most of the peptides, including the glycated and non-glycated were protected. The glycation sites not only influence the local region but also the distant area. The enhanced hydrogen protection after glycation suggests that the protein adopts a more stable conformation.

Structural changes of ultrasonicated bovine serum albumin revealed by hydrogen–deuterium exchange and mass spectrometry

The structural changes of bovine serum albumin (BSA) under high-intensity ultrasonication were investigated by fluorescence spectroscopy and mass spectrometry. Evidence for the ultrasonication-induced conformational changes of BSA was provided by the intensity changes and maximum-wavelength shift in fluorescence spectrometry. Matrix-assisted laser desorption–ionization time-of-flight mass spectroscopy (MALDI-TOF MS) revealed the increased intensity of the peak at the charge state +5 and a newly emerged peak at charge state +6, indicating that the protein became unfolded after ultrasonication. Prevalent unfolding of BSA after ultrasonication was revealed by hydrogen–deuterium exchange coupled with mass spectrometry (HDX-MS). Increased intensity and duration of ultrasonication further promoted the unfolding of the protein. The unfolding induced by ultrasonication goes through an intermediate state similar to that induced by a low concentration of denaturant.

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

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 β-lactoglobulin (β-LG). In this work, the conformational alteration of β-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 β-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 β-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.

A high throughput screening assay for identifying glycation inhibitors on MALDI-TOF target.

The Maillard reaction plays an important role in the food industry, however, the deleterious effects generated by the advanced glycation end-products (AGEs) have been well recognized. Many efforts have been made to seek new AGE inhibitors, in particular those natural ones without adverse effect. We have developed a rapid, mass spectrometry based, on-plate screening assay for novel AGE inhibitors. The glycation reaction, inhibition feedback as well as the subsequent MALDI mass spectrometric analysis occurred on one single MALDI plate. At 1:10 M ratio of peptide to sugar, as little as 4h incubation time allowed the screening test to be ready for analysis. DSP, inhibition and IC50 were calculated to evaluate selected inhibitors and resulting inhibition efficiencies were consistent with available references. We demonstrated that this method provide a potential high throughput screening assay to analyze and identify the anti-glycation agents.