田鼠巴贝虫可溶性虫体蛋白的质谱鉴定及生物信息学分析

Mass spectrometry identification and bioinformatics analysis of soluble proteins of Babesia microti

目的利用蛋白质组学及生物信息学方法对田鼠巴贝虫可溶性虫体蛋白进行分析。方法取田鼠巴贝虫阳性全血腹腔接种BALB/c小鼠,构建感染小鼠模型。取感染高峰期小鼠染虫血分离红细胞,Percoll密度梯度离心法富集田鼠巴贝虫虫体,冻融及超声法获得可溶性虫体蛋白。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定虫体蛋白相对分子质量分布,将含有可溶性虫体蛋白的凝胶按相对分子质量分为2个样品,采用电喷雾质谱鉴定法进行质谱鉴定。采集质谱鉴定数据,搜索Uniprot KB数据库中的巴贝虫、田鼠巴贝虫RI株和恶性疟原虫蛋白数据库。将鉴定到的蛋白与NCBI nr数据库中的蛋白序列进行比对,利用Blast2GO(Version2.8.0)中的Mapping功能对所有定量到的蛋白比对序列所关联的GO功能条目进行提取,用GO注释将鉴定到的虫体蛋白进行生物过程、分子功能和细胞组分分析。利用KAAS将目标蛋白序列与KEGG GENES数据库中的巴贝虫蛋白序列进行比对,通过同源/相似蛋白的KO号注释到相关KEGG通路上。结果Percoll梯度离心法获得富集后的田鼠巴贝虫虫体,通过冻融及超声法获得可溶性虫体蛋白,经SDS-PAGE鉴定发现有5条主带及7条次带。经质谱鉴定并与巴贝虫、田鼠巴贝虫RI株和恶性疟原虫蛋白数据库比对,分别鉴定到757、600和138个蛋白,其中独特肽段为2及以上的蛋白分别为368、375和12个。进一步分析发现,独特肽段数较多的蛋白为表面抗原或分泌抗原类蛋白、蛋白酶类、热休克家族蛋白及棒状体相关蛋白等。生物信息学分析,田鼠巴贝虫可溶性虫体蛋白按参与的生物过程共获得876条注释,按分子功能共获得219条注释,按细胞组分结果显示,共获得146条注释。通过同源/相似蛋白的KEGG注释,共提取到与可溶性虫体蛋白序列相关的172条KEGG信号/代谢通路。结论利用质谱鉴定及生物信息学方法分析田鼠巴贝虫可溶性虫体蛋白主要含有分泌蛋白、蛋白酶类和HSP家族成员蛋白。

Objective To analyze the soluble proteins of Babesia microti by using proteomics and bioinformatics methods. Methods BALB/c mice were intraperitoneally injected with 100 μl of blood containing B.microti parasites to establish the mouse model of B. microti infection. Red blood cells were separated from the B.microti-containing blood of the infected mice at the peak of infection. The B. microti parasites were enriched by Percoll density gradient centrifugation, freeze-thawed and sonicated to obtain soluble proteins of B. microti. SDS-PAGE was used to identify the molecular weight distribution of proteins. The gel containing soluble proteins was divided into 2 samples by molecular weight and identified by the electrospray ionization mass spectrometry（ESI-MS）method. The ESI-MS data were collected. Protein data for Babesia spp., B. microti（strain RI） and Plasmodium falciparum were searched on the Uniprot KB database. The identified proteins were aligned with the protein sequences in the NCBI nr database, and Gene Ontology（GO） function associated with the aligned sequences of all identified proteins were extracted with the Mapping function in Blast2 GO（Version 2.8.0）. The GO annotations were used for analysis of biological processes, molecular functions, and cellular components of the identified proteins. The target protein sequence was aligned with the Babesia protein sequence in the KEGG GENES database using KAAS,and the relevant KEGG pathway was annotated with the KO number of the homologous/similar protein.Results The B. microti parasites were enriched by Percoll gradient centrifugation and soluble proteins were obtained by freeze-thawing and sonication. SDS-PAGE showed that there were 5 major bands and 7 minor bands. After ESIMS and alignment with proteins of Babesia spp., B. microti（strain RI） and P. falciparum, 757, 600 and 138 proteins were identified, respectively, of which 368, 375 and 12 had more than 2 unique peptide segments,respectively. Further analysis revealed that those with more unique peptide segments were surface antigens or secreted antigenic proteins, proteases, heat shock family proteins and rod-like proteins. Bioinformatics analysis resulted in 876 annotations for the soluble proteins according to the biological processes in which they are involved, 219 annotations by molecular function and 146 annotations by cell components. By KEGG annotation of homologous/similar proteins, a total of 172 KEGG signal/metabolic pathways related to the soluble protein sequences were extracted. Conclusion The soluble proteins of B. microti are mainly composed of secretory proteins, proteases, and HSP family member proteins, as revealed by ESI-MS identification and bioinformatics analysis.