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首页> 外文期刊>Applied and Environmental Microbiology >Top-Down Proteomic Identification of Shiga Toxin 2 Subtypes from Shiga Toxin-Producing Escherichia coli by Matrix-Assisted Laser Desorption Ionization–Tandem Time of Flight Mass Spectrometry
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Top-Down Proteomic Identification of Shiga Toxin 2 Subtypes from Shiga Toxin-Producing Escherichia coli by Matrix-Assisted Laser Desorption Ionization–Tandem Time of Flight Mass Spectrometry

机译：通过基质辅助激光解吸电离-串联飞行时间质谱从上至下的蛋白质组学鉴定产志贺毒素的大肠杆菌中的志贺毒素2亚型。

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We have analyzed 26 Shiga toxin-producing Escherichia coli (STEC) strains for Shiga toxin 2 (Stx2) production using matrix-assisted laser desorption ionization (MALDI)–tandem time of flight (TOF-TOF) tandem mass spectrometry (MS/MS) and top-down proteomic analysis. STEC strains were induced to overexpress Stx2 by overnight culturing on solid agar supplemented with either ciprofloxacin or mitomycin C. Harvested cells were lysed by bead beating, and unfractionated bacterial cell lysates were ionized by MALDI. The A2 fragment of the A subunit and the mature B subunit of Stx2 were analyzed by MS/MS. Sequence-specific fragment ions were used to identify amino acid subtypes of Stx2 using top-down proteomic analysis using software developed in-house at the U.S. Department of Agriculture (USDA). Stx2 subtypes (a, c, d, f, and g) were identified on the basis of the mass of the A2 fragment and the B subunit as well as from their sequence-specific fragment ions by MS/MS (postsource decay). Top-down proteomic identification was in agreement with DNA sequencing of the full Stx2 operon (stx2) for all strains. Top-down results were also compared to a bioassay using a Vero-d2EGFP cell line. Our results suggest that top-down proteomic identification is a rapid, highly specific technique for distinguishing Stx2 subtypes.
机译：我们已经使用基质辅助激光解吸电离（MALDI）–串联飞行时间（TOF-TOF）串联质谱（MS / MS）分析了26种产生志贺毒素的大肠杆菌（STEC）菌株的志贺毒素2（Stx2）产生和自上而下的蛋白质组学分析。通过在补充有环丙沙星或丝裂霉素C的固体琼脂上过夜培养，诱导STEC菌株过表达Stx2。通过微珠敲打裂解收获的细胞，并通过MALDI将未分离的细菌细胞裂解物电离。通过MS / MS分析了Stx2的A亚基和成熟B亚基的A2片段。使用美国农业部（USDA）内部开发的软件，通过自上而下的蛋白质组学分析，使用序列特异性片段离子鉴定Stx2的氨基酸亚型。根据A2片段和B亚基的质量以及通过MS / MS从序列特异性片段离子中鉴定出Stx2亚型（a，c，d，f和g）（后源衰减）。自上而下的蛋白质组学鉴定与所有菌株的完整Stx2操纵子（ stx 2 ）的DNA测序一致。自上而下的结果也与使用Vero-d2EGFP细胞系的生物测定法进行了比较。我们的结果表明，自上而下的蛋白质组学鉴定是一种快速，高度特异性的技术，可用于区分Stx2亚型。

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