Current methodologies for proteomics of bacterial surface‐exposed and cell envelope proteins

Abstract

The study of surface-exposed proteins has received increasing attention following the advent of genomic sequencing, which in turn has enabled predictive tools and facilitated the technologies for their analysis by proteomics. The exterior topology of a bacterial pathogen is the interface between the cell and environment and thus is the initial mediator for infection, providing an important reservoir for components that may be used for novel vaccine development as well as the characterization of new drug targets. The study of such biological molecules has however, been under-represented in proteomics studies due to the difficulty involved in their analysis. Cell-envelope proteins in bacteria are typically difficult to characterize due to their low abundance, poor solubility, and the problematic isolation of pure surface fractions with only minimal contamination. Here, we describe different cell envelope preparations for proteomic characterization, focused principally on gel-free technologies. Fractionation techniques popularly used in proteomics are also explained with emphasis on surface and membrane-derived proteins/peptides. Conditional confirmation of localization is also explored with emphasis on different prediction algorithms as well as on analyses of surface peptide fractions by the use of different search programs and their implications for the unambiguous identification of surface-exposed and membrane-embedded proteins. Finally, different quantification techniques are discussed that are important for the validation of identifications and for highlighting novel proteins that may warrant further study by independent techniques.

Clostridium difficile secreted Pro‐Pro endopeptidase PPEP‐1 (ZMP1/CD2830) modulates adhesion through cleavage of the collagen binding protein CD2831

“…For this purpose, we performed in vitro cell surface ''shaving" experiments using C. difficile cells and recombinant PPEP-1 (rPPEP-1), in analogy to proteomic approaches which have been developed to study cell surface proteomes in general [26,27]. Following incubation of the C. difficile cells with rPPEP-1 for 1 h, cells were pelleted by centrifugation and the resulting supernatant analyzed for the presence of the CD2831 product peptide PAPPNTDEPIVNP.…”

Global relative and absolute quantitation in microbial proteomics

“…As the emergence and propagation of multiresistant strains, for example, of S. aureus, are a major threat in our health systems today [24], there is a strong need for novel antibiotic strategies. Granting access to potentially novel targets, extensive proteomic studies of S. aureus resolved more than 80% of the proteome in growing cells [19 ] and gained valuable information on the composition and changes of the surface proteome of staphylococci [25][26][27][28]. Quantitative proteomics is also pushed forward to in situ conditions by monitoring physiological changes of the pathogen after internalization by human host cells [29 ].…”

Properties of probiotic bacteria explored by proteomic approaches

“…[71,72] Shaving Technique that uses a proteolytic enzyme to digest the proteins exposed at the surface of bacteria. The resulting peptide mixtures are often analyzed using an LC-MS-MS approach [73] Proteome fractionation Essential step to simplify the sample's complexity before mass spectrometry analysis.…”