Improving Genome Annotation of Enterotoxigenic Escherichia coli TW10598 by a Label-Free Quantitative MS/MS Approach

The most commonly used genome annotation processes are to a great extent based on computational methods. However, those can only predict genes that have been described earlier or that have sequence signatures indicative of a gene function. We reported a synonymous proteogenomic approach for experimentally improving microbial genome annotation based on label-free quantitative MS/MS. The approach was exemplified by analysis of cell extracts from in vitro cultured enterotoxigenic Escherichia coli (ETEC) strain TW10598, as part of an effort to create a new reference ETEC genome sequence. The proteomic analysis yielded identification of 2,060 proteins, out of which 274 proteins were originally described as hypothetical. For 84% of the identified proteins we have provided description of their relative quantitative levels, among others, for 20 abundantly expressed ETEC virulence factors. Proteogenomic mapping supported the existence of four protein-coding genes that had not been annotated, and led to correction of translation start positions of another nine.

当前最常用的基因组注释（genome annotation）流程，在很大程度上基于计算方法。但此类方法仅能预测此前已有记载的基因，或具备指示基因功能的序列特征的基因。本研究报道了一种基于无标记定量串联质谱（MS/MS）的同义蛋白质基因组学（proteogenomic）方法，用于实验层面优化微生物基因组注释。作为构建新型产肠毒素大肠杆菌（enterotoxigenic Escherichia coli, ETEC）参考基因组序列工作的一部分，该方法通过分析体外培养的产肠毒素大肠杆菌TW10598菌株的细胞提取物得以实例验证。本次蛋白质组学分析共鉴定出2060种蛋白质，其中274种最初被标注为假设蛋白（hypothetical proteins）。针对84%的已鉴定蛋白质，我们提供了其相对定量水平的相关信息，其中包括20种高表达的ETEC毒力因子。蛋白质基因组学图谱分析证实了4个此前未被注释的蛋白质编码基因（protein-coding genes）的存在，并修正了另外9个基因的翻译起始位点（translation start positions）。