Assessment of protoxin composition of Bacillus thuringiensis strains by use of polyacrylamide gel block and mass spectrometry

Abstract

Assessment of protoxin composition in Bacillus thuringiensis parasporal crystals is principally hampered by the fact that protoxins in a single strain usually possess high sequence homology. Therefore, new strategies towards the identification of protoxins have been developed. Here, we established a powerful method through embedding solubilized protoxins in a polyacrylamide gel block coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of in-gel-generated peptides for protoxin identification. Our model study revealed that four protoxins (Cry1Aa, Cry1Ab, Cry1Ac and Cry2Aa) and six protoxins (Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa, Cyt1Aa, and Cyt2Ba) could be rapidly identified from B. thuringiensis subsp. kurstaki HD1 and subsp. israelensis 4Q2-72, respectively. The experimental results indicated that our method is a straightforward tool for analyzing protoxin expression profile in B. thuringiensis strains. Given its technical simplicity and sensitivity, our method might facilitate the present screening program for B. thuringiensis strains with new insecticidal properties.

Proteomic Analysis of Bacillus thuringiensis at Different Growth Phases by Using an Automated Online Two-Dimensional Liquid Chromatography-Tandem Mass Spectrometry Strategy

“…Cells harvested at different time points (10,20, and 32 h) were washed three times with a suspension in previously chilled phosphate-buffered saline (10 mM [pH 7.8]) and placed in a prechilled pestle. Liquid nitrogen was scooped on top of the cells to flash freeze them.…”

“…Thus, the eluted peptides were trapped rotatorily in two enrichment columns by a 12-stepelution from the SCX column, and the peptides eluted from the previous run were further separated by an RP column, followed by MS/MS analysis. 20-min gradient in 80% buffer B, and 5 min in 80% buffer B, a 1-min gradient in 100% buffer A, and a 12-min reequilibration in 100% buffer A. The flow rate for the reversed-phase separation was maintained at 200 nl/min after splitting.…”

Isolation of novel Bacillus species showing high mosquitocidal activity against several mosquito species

“…VB17 produced two major proteins bands of about 180 and 160 kDa whereas VB24 produced a thick protein band of about 130 kDa (data not shown). To determine the protein composition in the sporulated cultures of these Bacillus isolates, the solubilized protein mixture from each isolate was analyzed by use of polyacrylamide gel block coupled with LC-MS/MS as described previously with minor modifications at the Protein Chemistry Core Laboratory, Interdisciplinary Center for Biotechnology Research, University of Florida (Gainesville, FL) (Fu et al, 2008;Golovko and Murphy, 2008). The proteins of VB17 and VB24 showed 100% sequence identity with spore coat protein, CotZ of a marine Bacillus sp.…”

Identification and Characterization of Three Previously Undescribed Crystal Proteins from Bacillus thuringiensis subsp. jegathesan

“…Recently, we reported a convenient and straightforward method for analyzing the overall protoxin composition in B. thuringiensis strains by using a polyacrylamide gel block coupled to liquid chromatographytandem mass spectrometry (LC-MS/MS) (18). This improved method had advantages in terms of accuracy and efficiency over traditional protein-based means, as well as one-dimensional SDS-PAGE coupled to an MS technique, when analyzing protoxins with high sequence homology produced in a single strain (18)(19)(20). During the search for new toxins from various B. thuringiensis strains using this improved MS method, we analyzed the protoxin complement in the parasporal body of B. thuringiensis subsp.…”