超速离心和酸沉淀制备牛乳清的蛋白质组学研究

Proteomics Study on Preparation of Bovine Whey by Ultracentrifugation and Acid Precipitation

本研究旨在对超速离心法和酸沉淀法鉴定的乳清蛋白进行分析,并比较不同样品制备方法对乳清蛋白组鉴定结果的影响。采集牛奶样品去除脂肪,利用超速离心和酸沉淀法制备乳清蛋白,液相色谱串联质谱分析结合数据库搜索鉴定,比较两种方法制备的乳清蛋白的差异,分析了差异蛋白参与的生物过程、细胞组成和分子功能等。聚类分析和主成分分析展示了两种样品制备方法的差异蛋白,超速离心方法鉴定的表达量增加的蛋白有53个,包括β-酪蛋白、糖基化依赖细胞黏附分子1和多聚免疫球蛋白受体等;酸沉淀方法鉴定的表达量增加的蛋白有21个,包括CD9抗原、β2-微球蛋白和β-乳球蛋白等。将两种方法获得的差异蛋白根据蛋白注释分类发现,超速离心法中高表达的蛋白主要位于胞外区域、囊泡、内膜系统、内质网和高尔基体等,涉及蛋白结合、酶调节活性、受体结合和抗氧化活性等;酸沉淀法中高表达的蛋白主要位于胞外区域和囊泡等,涉及结合、核酸酶活性和离子结合等。结果提示,两种方法鉴定的蛋白质组表达模式存在差异,乳清蛋白样品制备方法的结合可以获得更多的鉴定蛋白,为乳清蛋白组学研究中样品制备方法的选择提供参考依据。

This study was aimed to analyze whey proteins identified by ultracentrifugation and acid precipitation,and compare the effects of different sample preparation methods on the identification results of whey proteome.Milk samples were collected to remove fat.Whey protein was prepared by ultracentrifugation and acid precipitation,the difference between two methods was compared by LC-MS and database search,the biological processes,cell composition and molecular functions of different proteins were analyzed.Cluster analysis and principal component analysis showed that 53 proteins with increased expression levels were identified by ultracentrifugation,includingβ-casein,glycosylation-dependent cell adhesion molecule 1 and polyimmunoglobulin receptors;21 proteins with increased expression levels were identified by acid precipitation method,including CD9 antigen,β2-microglobulin andβ-lactoglobulin.According to annotation classification,proteins highly expressed in ultracentrifugation were mainly located in extracellular regions,vesicles,intimal system,endoplasmic reticulum and Golgi apparatus,involving protein binding,enzyme-modulating activity,receptor binding and resistance;Proteins highly expressed in acid precipitation were also mainly located in extracellular regions and vesicles,involving binding,nuclease activity,and ionic binding.Based on the above analysis results,the proteome profiles identified by two methods were different,and the combination of two methods could obtain more identified proteins.This study could provide a basis for the selection of sample preparation methods in whey proteomics research.