Cell Shaving and False-Positive Control Strategies Coupled to Novel Statistical Tools to Profile Gram-Positive Bacterial Surface Proteomes.
A powerful start to the discovery and design of novel vaccines, and for better understanding of host-pathogen interactions, is to profile bacterial surfaces using the proteolytic digestion of surface-exposed proteins under mild conditions. This "cell shaving" approach has the benefit of both identifying surface proteins and their surface-exposed epitopes, which are those most likely to interact with host cells and/or the immune system, providing a comprehensive overview of bacterial cell topography. An essential requirement for successful cell shaving is to account for (or minimize) cellular lysis that can occur during the shaving procedure and thus generate data that is biased towards non-surface (e.g., cytoplasmic) proteins. This is further complicated by the presence of "moonlighting" proteins, which are proteins predicted to be intracellular but with validated surface or extracellular functions. Here, we describe an optimized cell shaving protocol for Gram-positive bacteria that uses proteolytic digestion and a "false-positive" control to reduce the number of intracellular contaminants in these datasets. Released surface-exposed peptides are analyzed by liquid chromatography (LC) coupled to high-resolution tandem mass spectrometry (MS/MS). Additionally, the probabilities of proteins being surface exposed can be further calculated by applying novel statistical tools.
School of Molecular Bioscience, The University of Sydney, Sydney, NSW, Australia.|Department of Oral Biological and Medical Sciences, Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada.
Nestor
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Solis
School of Molecular Bioscience, The University of Sydney, Sydney, NSW, Australia. stuart.cordwell@sydney.edu.au.|Discipline of Pathology, School of Medical Sciences, The University of Sydney, The Hub Building D17, Sydney, NSW, 2006, Australia. stuart.cordwell@sydney.edu.au.|Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia. stuart.cordwell@sydney.edu.au.
Stuart J
SJ
Cordwell
D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry; D002853:Chromatography, Liquid; D018604:Epitope Mapping; D000939:Epitopes / Q000378:metabolism; D006094:Gram-Positive Bacteria / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
D001426:Bacterial Proteins; D000939:Epitopes; D008565:Membrane Proteins
Cell shaving; Gram-positive bacteria; Mass spectrometry; Surface proteomics; Surfaceome