"One-Pot" Sample Processing Method for Proteome-Wide Analysis of Microbial Cells and Spores

Proteomics. Clinical Applications
Bhagyashree Nandakishor SwargeLeo J de Koning

Abstract

Bacterial endospores, the transmissible forms of pathogenic bacilli and clostridia, are heterogeneous multilayered structures composed of proteins. These proteins protect the spores against a variety of stresses, thus helping spore survival, and assist in germination, by interacting with the environment to form vegetative cells. Owing to the complexity, insolubility, and dynamic nature of spore proteins, it has been difficult to obtain their comprehensive protein profiles. The intact spores of Bacillus subtilis, Bacillus cereus, and Peptoclostridium difficile and their vegetative counterparts were disrupted by bead beating in 6 m urea under reductive conditions. The heterogeneous mixture was then double digested with LysC and trypsin. Next, the peptide mixture was pre-fractionated with zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) followed by reverse-phase LC-FT-MS analysis of the fractions. "One-pot" method is a simple, robust method that yields identification of >1000 proteins with high confidence, across all spore layers from B. subtilis, B. cereus, and P. difficile. This method can be employed for proteome-wide analysis of non-spore-forming as well as spore-forming pathogens. Analysis of spore prote...Continue Reading

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Datasets Mentioned

BETA
PXD008242

Methods Mentioned

BETA
Protein Assay
deamination
GTPase

Software Mentioned

ProtParam
MASCOT
DAVID
MASCOT DISTILLER
MASCOT MudPIT
MDRO
DBToolkit
POPI
MudPIT

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