Department for Bioanalysis and Horizon Technologies, Health Protection Agency Centre for Infections, 61 Colindale Avenue, London NW9 5EQ, UK.
BMC Microbiol. 2010 Feb 11;10:44. doi: 10.1186/1471-2180-10-44.
Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major cause of human gastroenteritis worldwide. The outer membrane proteins expressed by S. Typhimurium mediate the process of adhesion and internalisation within the intestinal epithelium of the host thus influencing the progression of disease. Since the outer membrane proteins are surface-exposed, they provide attractive targets for the development of improved antimicrobial agents and vaccines. Various techniques have been developed for their characterisation, but issues such as carryover of cytosolic proteins still remain a problem. In this study we attempted to characterise the surface proteome of S. Typhimurium using Lipid-based Protein Immobilisation technology in the form of LPI FlowCells. No detergents are required and no sample clean up is needed prior to downstream analysis. The immobilised proteins can be digested with proteases in multiple steps to increase sequence coverage, and the peptides eluted can be characterised directly by liquid chromatography - tandem mass spectrometry (LC-MS/MS) and identified from mass spectral database searches.
In this study, 54 outer membrane proteins, were identified with two or more peptide hits using a multi-step digest approach. Out of these 28 were lipoproteins, nine were involved in transport and three with enzyme activity These included the transporters BtuB which is responsible for the uptake of vitamin B12, LamB which is involved in the uptake of maltose and maltodextrins and LolB which is involved in the incorporation of lipoproteins in the outer membrane. Other proteins identified included the enzymes MltC which may play a role in cell elongation and division and NlpD which is involved in catabolic processes in cell wall formation as well as proteins involved in virulence such as Lpp1, Lpp2 and OmpX.
Using a multi-step digest approach the LPI technique enables the incorporation of a multi-step protease work flow ensuring enough sequence coverage of membrane proteins subsequently leading to the identification of more membrane proteins with higher confidence. Compared to current sub-cellular fractionation procedures and previous published work, the LPI technique currently provides the widest coverage of outer membrane proteins identified as demonstrated here for Salmonella Typhimurium.
肠炎沙门氏菌血清型 Typhimurium（S. Typhimurium）是全球人类肠胃炎的主要病因。S. Typhimurium 表达的外膜蛋白介导了在宿主肠道上皮细胞中的黏附和内化过程，从而影响疾病的进展。由于外膜蛋白是表面暴露的，因此它们为开发改良的抗菌剂和疫苗提供了有吸引力的靶标。已经开发了各种技术来对其进行表征，但仍存在诸如胞质蛋白残留等问题。在这项研究中，我们尝试使用脂质蛋白固定化技术（LPI）以 LPI FlowCells 的形式来表征 S. Typhimurium 的表面蛋白质组。不需要去污剂，也不需要在下游分析之前对样品进行清洗。固定化的蛋白质可以用蛋白酶进行多次消化以增加序列覆盖率，洗脱的肽可以直接通过液相色谱 - 串联质谱（LC-MS/MS）进行表征，并从质谱数据库搜索中进行鉴定。
在这项研究中，使用多步消化方法，通过两个或更多肽命中鉴定出 54 种外膜蛋白。其中 28 种是脂蛋白，9 种参与运输，3 种具有酶活性。这些包括负责摄取维生素 B12 的转运体 BtuB、参与麦芽糖和麦芽糊精摄取的 LamB 以及参与脂蛋白掺入外膜的 LolB。鉴定出的其他蛋白质包括可能在细胞伸长和分裂中发挥作用的 MltC 酶和参与细胞壁形成的分解代谢过程的 NlpD 以及参与毒力的蛋白质，如 Lpp1、Lpp2 和 OmpX。
使用多步消化方法，LPI 技术能够整合多步蛋白酶工作流程，确保膜蛋白的序列覆盖率足够高，从而更有信心地鉴定出更多的膜蛋白。与当前的亚细胞分级分离程序和以前发表的工作相比，LPI 技术目前提供了最广泛的外膜蛋白覆盖范围，如本文所示，用于肠炎沙门氏菌 Typhimurium。
