Bacterial Protein Profiling——Comparison of Three Mass Spectrometry Methodologies

细菌的蛋白质组成分析——深入比较三种质谱方法

Profiling the protein composition of bacteria is essential for understanding their biology,physiology and interaction with environment.Mass spectrometry has become a pivotal tool for protein analysis,facilitating the examination of expression levels,molecular masses and structural modifications.In this study,we compared the performance of three widely-used mass spectrometry methods,i.e.,matrix-assisted laser desorption/ionization(MALDI)protein fingerprinting,top-down proteomics and bottom-up proteomics,in the profiling of bacterial protein composition.It was revealed that bottom-up proteomics provided the highest protein coverage and exhibited the greatest protein profile overlap between bacterial species.In contrast,MALDI protein fingerprinting demonstrated superior detection reproducibility and effectiveness in distinguishing between bacterial species.Although top-down proteomics identified fewer proteins than bottom-up approach,it complemented MALDI fingerprinting in the discovery of bacterial protein markers,both favoring abundant,stable,and hydrophilic bacterial ribosomal proteins.This study represents the most systematic and comprehensive comparison of mass spectrometry-based protein profiling methodologies to date.It provides valuable guidelines for the selection of appropriate profiling strategies for specific analytical purposes.This will facilitate studies across various fields,including infection diagnosis,antimicrobial resistance detection and pharmaceutical target discovery.

细菌蛋白质组成分析对于了解其生物学、生理学及其与环境的相互作用至关重要.质谱是用于蛋白质分析的最有力工具之一,在蛋白质的分子量确定、表达水平测量及结构修饰分析等研究中不可或缺.本文比较了蛋白质指纹质谱、自上而下蛋白质组学和自下而上蛋白质组学等3种广泛使用的质谱方法在细菌蛋白成分分析中的表现.结果表明,自下而上蛋白质组学提供了最高的蛋白覆盖率,同时也在不同菌种之间显示出最大的蛋白质图谱重合度.相比之下,蛋白质指纹质谱显示出最高的检测再现性以及菌种区分或鉴定的高效性.自上而下蛋白组学检测到的细菌蛋白质数量明显少于自下而上蛋白质组学,但其可以与蛋白质指纹质谱兼容(二者均偏向于检测高丰度、高稳定和高亲水性的核糖体蛋白质),在菌种鉴定的同时为蛋白标志物的发现提供重要手段.本文对基于质谱的蛋白质组成分析方法进行了比较,为特定分析目标的方法选择提供了指导意见.这将对于细菌感染诊断、抗生素耐受性分析和抗生素作用靶点发现等多个领域的研究具有重要价值.