基于串联质谱标签法和平行反应监测技术的氟喹诺酮耐药沙门菌蛋白质组学分析

【背景】随着沙门菌对氟喹诺酮类药物的耐药性不断增强,对其耐药机理的研究显得尤为迫切和重要,蛋白质组学分析将为沙门菌的耐药机理研究提供新的靶点和方向。【目的】对鼠伤寒沙门菌诱导获得耐药性前后进行蛋白质组学分析,为深入研究沙门菌耐药机理奠定基础。【方法】用环丙沙星对鼠伤寒沙门菌ATCC13311进行耐药性诱导,利用串联质谱标签法(Tandem mass tag,TMT)对其耐药性进行差异蛋白的筛选和生物信息学分析,并选取15个差异蛋白进行平行反应监测(Parallel reaction monitoring,PRM)靶向蛋白验证。【结果】筛选出318个差异表达蛋白,其中上调159个,下调159个,涉及的KEGG通路主要包括细菌趋药性、ABC转运蛋白、双组分系统等;PRM定量到13个验证蛋白且变化趋势与TMT一致。【结论】通过TMT定量结合PRM靶向验证对鼠伤寒沙门菌耐药前后进行蛋白质组学分析,筛选出多个差异蛋白和代谢通路,包括外排泵相关蛋白、外膜蛋白、双组分相关蛋白及通路、细菌趋化性相关蛋白及通路等,为沙门菌氟喹诺酮类耐药机理的深入研究奠定了基础。

Fluoroquinolone resistant Salmonella proteomics analysis based on tandem mass tag and Parallel reaction monitoring techniques

Background] As Salmonella resistance to fluoroquinolones has increased gradually, it is very urgent and important to study the mechanism of drug resistance. Proteomics analysis will provide new targets and directions for the study on resistance mechanism in Salmonella. Objective] Proteomic analysis of Salmonella typhimurium was performed before and after induction, in order to lay the foundation for further studies on the mechanism in Salmonella resistance. Methods] Ciprofloxacin was used to induce Salmonella typhimurium ATCC13311 to obtain resistance, screening and bioinformatics analysis of differential proteins were performed by using tandem mass tag (TMT). Besides, fifteen differential proteins were selected for (parallel reaction monitoring PRM) target verification. Results] The results showed that a total of 318 differentially expressed proteins were screened, among which 159 proteins were up-regulated and 159 proteins were down-regulated. The KEGG pathways involved in these proteins mainly including bacterial chemotaxis, ABC transporters, two-component systems, etc; PRM was successfully quantitated to 13 validated proteins and the trend was consistent with TMT. Conclusion] In this study, the proteomics analysis of Salmonella typhimurium parental strain and ciprofloxacin-resistant strain was conducted by TMT quantitatively combined with PRM target verification, screening out a variety of differential proteins and metabolic pathways, including efflux pump related proteins, outer membrane proteins, two-component related proteins and pathways, bacteria chemotaxis related proteins and pathways, etc. The study laid a certain foundation for further studies on the mechanism of fluoroquinolone resistance in Salmonella.