Background. 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. Results. 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. Conclusion. 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.
Bibliographical noteFunding Information:
We would like to acknowledge the proteomic expertise at the Proteomics Core Facility at the University of Gothenburg and the in-house data evaluation performed by Max Davidson at Nanoxis AB, Gothenburg, Sweden. This project was funded by the Health Protection agency PhD studentship.