基于蛋白质组学和时空动态的微波促进蛋白质糖基化反应的机制研究

Previous research indicated that microwave could promote the glycation reaction of the protein，and the reaction degree of each site is highly related with protein structure and the non-uniform temperature distribution of microwave heating. However, the promotion mechanism is yet unknown. According to the scientific problem, from the perspective of proteomics and space-time dynamics change during microwave processing, this project aims to investigate the promoted mechanism of microwave on the glycation reaction between ovalbumin and glucose under different time and space by establishing cylindrical dry and wet model. The temperature distribution of microwave heating sample was determined by fluorescence optical fiber temperature sensor with no contact mini probe and infrared thermographic imager. After transverse and longitudinal split of sample, the glycation reaction process under different space and time is supervised by studying the reaction degree and physico-chemical properties of products through electrophoresis, chromaticity, differential scanning calorimetry (DSC), and spectrum analysis. The reaction site, precise identification and relative quantification of glycation products are analyzed by means of proteomics, such as digestion in gel, LC FTICR MS, ETD-MSMS, 18O stable isotope labeling, et al. The rule of glycation reaction is revealed in microwave field. The relationship among microwave conditions, temperature distribution, and glycation reaction is established under different space and time. Then the mechanism of microwave promoting glycation reaction of protein based on proteomics and space-time dynamics. This research can provide theoretical basis for the directional control of protein glycation reaction in microwave field. And it may play a profound role in the development and progressing in food science.

前期研究发现，微波会促进蛋白质的糖基化反应，且反应位点、反应程度与蛋白质结构及微波加热不均匀性密切相关，但是其促进机制尚不清楚。针对这一科学问题，本项目拟从蛋白质组学和时空动态变化角度出发，以卵清蛋白和葡萄糖为对象，建立干样和湿样的圆柱模型，采用荧光光纤温度传感器的无接触小型探针和红外热像仪监测其温度分布；通过样品分割、电泳、色度、DSC、光谱技术等分别从反应程度和理化性质方面监测反应历程；采用胶上酶解、LC FTICR MS、ETD MSMS、18氧稳定同位素标记等方法对蛋白质糖基化产物的反应位点进行精确定位和相对定量分析；揭示微波场内蛋白质糖基化反应的规律，建立不同时空条件下微波条件-温度分布-糖基化反应之间的关系，从分子水平和时空动态变化方面阐述微波促进蛋白质糖基化反应的内在机制。本项目成果为实现微波场中蛋白质糖基化反应的定向调控奠定理论基础，对食品科学的进步与发展具有重要意义。

项目以卵清蛋白（OVA）和葡萄糖混合物建立反应体系，首先采用生物质谱方法研究发现冷冻干燥过程中蛋白质上的赖氨酸易发生糖基化修饰，其中K62和K264是最活泼的糖基化位点，这些糖基化位点主要分布于OVA分子的表面，而疏水袋和蛋白质内部的6条β-折叠片上不会发生糖基化修饰；在微波场内，OVA和葡萄糖反应体系存在3个反应热点，在反应热点，反应模型的温度会显著升高，糖基化修饰程度显著增大，同时产生大量的OVA糖基化中高级产物，而在非反应热点也会发生糖基化反应，以糖基化初级产物为主，说明短时微波加热会加快OVA糖基化反应的历程；不同微波场作用的糖基化OVA产物的功能特性、消化性和功能性基团均有显著差异，过敏性降低；通过生物质谱进一步分析发现在反应热点和非热点生成的OVA糖基化产物中糖基化位点数量无显著性差异，但是反应热点有更多的精氨酸被糖基化修饰，赖氨酸发生更多的脱水脱氧等衍生反应，且存在交联物质；通过进一步的三维结构分析和比对，我们发现微波加热导致的糖基化修饰位点主要位于OVA球蛋白的表面，而位于OVA结构中β-折叠片上的K和R均未发生糖基化修饰，可能是由于微波效应不能使OVA分子内部产生热量，而更多的集中于分子表面产生大量的热量，从而促进OVA球蛋白表面的氨基酸发生糖基化修饰。这一发现为球蛋白的表面修饰和新型表面活性剂的研发提供了新途径和新方法。