Ionic liquid-assisted sample preparation mediates sensitive proteomic analysis of Bacillus subtilis spores

Endospore-forming bacteria are ubiquitous. Bacterial endospores are multilayered proteinaceous structures that protects the bacterial genome during stress conditions. They are also responsible for a wide range of critical clinical infections in humans. Precise analysis of spore-forming pathogens remains a major challenge in the field of proteomics because spore structures are highly resistant to conventional solubilizers and denaturing agents, such as sodium dodecyl sulfate and urea. We present an ionic liquid-assisted (i-soln) technique of sample preparation, called pTRUST, which enables shotgun analysis of Bacillus subtilis spores even when the starting materials are in the sub-microgram range. In proteomic analysis, this technique shows 50-2000-fold higher sensitivity than other conventional gel-based or gel-free methods (including one-pot sample processing). Using this technique, we identified 445 proteins with high confidence from trace amounts of highly pure spore preparations, including 52 of the 79 proteins (approximately 70%) previously demonstrated to be localized in spores in the SubtiWiki database and detected through direct protein analysis. Consequently, 393 additional proteins were identified as candidates for spore constitutive proteins. Twenty of these newly identified candidates were produced as green fluorescent protein fusion proteins, and each was evaluated for authenticity as a spore constituent using fluorescence microscopy analysis. The pTRUST method's sensitivity and reliability using the i-soln system, together with hitherto unreported proteins in spores, will enable an array of spore research for biological and clinical applications.