Fagerquist Clifton K, Sultan Omar
Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 800 Buchanan Street, Albany, CA 94710, USA.
J Biomed Biotechnol. 2010;2010:123460. doi: 10.1155/2010/123460. Epub 2011 Feb 10.
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.
已开发出一种方法，使用基质辅助激光解吸/电离飞行时间串联质谱（MALDI-TOF-TOF-MS/MS）和基于内部开发的网络软件的自上而下蛋白质组学，从大肠杆菌O157:H7中鉴定志贺毒素2（α-Stx2）的α亚基。通过在添加了引发细菌SOS反应的抗生素的固体琼脂上培养大肠杆菌O157:H7来诱导Stx2的表达。细菌细胞裂解物在人源酶弗林蛋白酶的存在下孵育，该酶将α-Stx2切割成A1（约28 kDa）和A2（约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/二硫键还原样品的MALDI-TOF-MS在质荷比（m/z）5286处显示出一个峰，对应于A2片段。尽管通过自下而上蛋白质组学证实了A1片段的存在，但未观察到与之对应的峰。通过MALDI-TOF-TOF-MS/MS和自上而下蛋白质组学明确鉴定出m/z 5286处的峰为α-Stx2的A2片段。
