纳升级反相液相色谱-串联质谱法分析麦氏弧菌蛋白质组  

Proteomic Analysis of Vibrio Metschnikovii from Homarus americanus by Nanoflow Reversed Phase Liquid Chromatography-tandem Mass Spectrometry

作　　者：李正义[1] 贾俊涛[1] 姜英辉[1] 王骏[1] 罗继 崔淑华[1] 徐彪[1] 梁成珠[1] 张铁军[1] 

机构地区：[1]山东出入境检验检疫局检验检疫技术中心,山东青岛266002 [2]ABSCIEX公司上海应用实验室,上海200233

出　　处：《安徽农业科学》2015年第33期156-158,183,共4页Journal of Anhui Agricultural Sciences

基　　金：国家质检总局科技项目(2012IK305;2013IK196;2015IK203)

摘　　要：[目的]较系统地研究分析麦氏弧菌提取蛋白质。[方法]利用SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和纳升级反相液相色谱-串联质谱(nano-RPLC-MS/MS)法系统分析了来源于美国龙虾的一株麦氏弧菌F5-1提取总蛋白和总蛋白直接酶解产物,并进行数据库检索鉴定蛋白质。纳升级反相液相色谱分析条件为:上样量为5μl,经C18预柱(200μm×0.5 mm,3μm,120)脱盐10 min,再经过Chrom XP C_(18)-CL毛细管柱(75μm×15 cm,3μm,120)分析,2%乙腈(0.1%甲酸)-98%乙腈(0.1%甲酸)梯度洗脱90 min,采用ESI-Q-TOF MS,在IDA采集模式下分析鉴定麦氏弧菌提取总蛋白酶解产物。[结果]直接酶解鉴定到1 538种蛋白质,通过Gene Ontology分析了麦氏弧菌F5-1提取总蛋白的分子功能、生物进程和细胞组分。提取的麦氏弧菌蛋白具有催化、结合、结构分子、转录调节因子等功能活性,其中催化活性的蛋白比例较大,且多为参与代谢进程的相关酶类。[结论]研究可为麦氏弧菌蛋白质组学的深入研究奠定基础。[Objective] To systematically study and analyze protein extraction of Vibrio metschnikovii. [Method] SDS-PAGE coupled with nanoflow reversed phase liquid chromatography-tandem mass spectrometry（ nano-RPLC-MS / MS） was developed for analyzing extracted proteins and extracted proteins of gel-free digestion products from Vibrio metschnikovii F5-1 isolated from Homarus americanus. All extracted proteins were searched and identified in accordance with the Uniprot database database. The digested peptides were desalted using a C18pre-column（ 200 μm × 0. 5 mm,3 μm,120 ）. Nano LC separation was performed on a Chrom XP C18-CL capillary column（ 75 μm × 15 cm,3μm,120 ） using a gradient elution program of 2% acetonitrile solution（ containing 0. 1% formic acid） and 98% acetonitrile solution（ containing 0. 1% formic acid） as the mobile phase with a flow rate of 300 nl / min and the injection volume of 5 μl. Quadruple-time-of-flight mass spectrometer（ Q-TOF-MS） and electrospray ion source（ ESI） were applied for qualitative analysis under the information dependent acquisition（ IDA） mode. [Result]1 538 kinds of proteins were identified by gel-free digestion method. Based on the Gene Ontology analysis,the extracted proteins identified for Vibrio metschnikovii F5-1 were differentially displayed according to molecular functions,biological process and cellular component. The function of proteins included catalytic activity,binding activity,structural molecular activity and translation regulator activity,in which catalytic activity was the major molecular functions involved in the metabolic process. [Conclusion]The study can lay a foundation for deep research of Vibrio metschnikovii proteomics.

关 键 词：纳升及反相液相色谱-串联质谱 麦氏弧菌 蛋白质组 

分 类 号：S182[农业科学—农业基础科学]