Gel-free proteomic identification of the Bacillus subtilis insoluble spore coat protein fraction

Species from the genus Bacillus have the ability to form endospores, dormant cellular forms
that are able to survive heat and acid preservation techniques commonly used in the food
industry. Resistance characteristics of spores towards various environmental stresses are in
part attributed to their coat proteins. Previously, 70 proteins have been assigned to the spore
coat of Bacillus subtilis using SDS-PAGE, 2-DE gel approaches, protein localization studies
and genome-wide transcriptome studies. Here, we present a ‘‘gel-free’’ protocol that is
capable of comprehensive B. subtilis spore coat protein extraction and addresses the insoluble
coat fraction. Using LC-MS/MS we identified 55 proteins from the insoluble B. subtilis spore
coat protein fraction, of which 21 are putative novel spore coat proteins not assigned to the
spore coat until now. Identification of spore coat proteins from a B. subtilis food-spoilage
isolate corroborated a generic and "applied" use of our protocol. To develop specific and
sensitive spore detection and/or purification systems from food stuff or patient material,
suitable protein targets can be derived from our proteomic approach. Finally, the protocol
can be extended to study cross-linking among the spore coat proteins as well as for their
quantification.