Top-down proteomic identification of furin-cleaved α-subunit of Shiga toxin 2 from Escherichia coli O157:H7 using MALDI-TOF-TOF-MS/MS.
Top-down proteomic identification of furin-cleaved α-subunit of Shiga toxin 2 from Escherichia coli O157:H7 using MALDI-TOF-TOF-MS/MS.
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DOI：
10.1155/2010/123460
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发表时间：
2010
影响因子：
--
通讯作者：
Sultan O
中科院分区：
文献类型：
--
作者：
Fagerquist CK;Sultan O
采用基质辅助激光解吸电离飞行时间-飞行时间串联质谱（MALDI-TOF-TOF-MS/MS）和自顶向下蛋白质组学技术，建立了一种鉴定大肠杆菌O 157：H7中志贺滋贺2 α亚基（α-Stx 2）的方法。通过培养E. coli O 157：H7在添加有增强细菌SOS反应的抗生素的固体琼脂上的表达。将细菌细胞裂解物在存在弗林蛋白酶的情况下孵育，弗林蛋白酶是一种人酶，可将α-Stx 2切割成A1（~28 kDa）和A2（~5 kDa）蛋白片段。随后的二硫键还原步骤使A1与A2分离。 弗林蛋白酶消化/二硫化物还原样品的MALDI-TOF-MS显示质荷比（m/z）5286处的峰，对应于A2片段。 没有观察到对应于A1片段的峰，尽管其存在通过自下而上的蛋白质组学证实。 通过MALDI-TOF-TOF-MS/MS和自顶向下蛋白质组学确定m/z 5286处的峰为α-Stx 2的A2片段。
A method has been developed to identify the α-subunit of Shiga toxin 2 (α-Stx2) from Escherichia coli O157:H7 using matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomics using web-based software developed in-house. Expression of Stx2 was induced by culturing E. coli O157:H7 on solid agar supplemented with an antibiotic that elicits the bacterial SOS-response. Bacterial cell lysates were incubated in the presence of furin, a human enzyme, that cleaves α-Stx2 into A1 (~28 kDa) and A2 (~5 kDa) protein fragments. A subsequent disulfide reduction step unlinked A1 from A2. MALDI-TOF-MS of the furin-digested/disulfide-reduced sample showed a peak at mass-to-charge (m/z) 5286 that corresponded to the A2 fragment. No peak was observed that corresponded to the A1 fragment although its presence was confirmed by bottom-up proteomics. The peak at m/z 5286 was definitively identified by MALDI-TOF-TOF-MS/MS and top-down proteomics as the A2 fragment of α-Stx2.
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影响因子：
7.4
作者：
Fagerquist, CK;Miller, WG;Mandrell, RE
通讯作者：
Mandrell, RE
影响因子：
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影响因子：
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影响因子：
7.4
作者：
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