Complete sequences of small acid‐soluble proteins from Bacillus globigii

Abstract

Three abundant small acid-soluble proteins (SASPs) from spores of Bacillus globigii were sequenced using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with post-source decay and nanoelectrospray collision-induced dissociation tandem mass spectrometry. The proteins were extracted from spores with 1 M HCl. Scanning electron micrographs of spores before and after acid extraction show that the spores retain their overall structure but have a shriveled texture following the acid treatment. Extracted SASPs were purified by high-performance liquid chromatography and molecular masses of the SASPs were identified at 7068 (SASP-1), 7332 (SASP-2), and 8889 (gamma-SASP). De novo peptide sequencing was used to determine the protein sequences. The correct ordering of peptide sequences was aided by mapping overlapping enzymatic digests and by comparison with homologous SASPs from Bacillus stearothermophilus. B. globigii is used in many field tests as a surrogate for B. anthracis. Thus complete SASP sequences from B. globigii will facilitate the development of methods for rapid identification of bacteria based on mass spectrometry and the examination of taxonomic relationships between Bacillus species.

Identification of Bacillus anthracis by Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and Artificial Neural Networks

“…On the other hand, the presence of strong SASP signals in mass spectra of Bacillus cultures may be the result of the specifically increased sensitivity of the TFA sample preparation technique for these markers. For example, it was reported earlier that acid treatment effectively extracts SASPs from spore preparations (31,37). Second, SASPs are easy ionizable and are probably preferentially detected by MALDI-TOF MS.…”

Rapid characterization of Bacillus spores targeting species‐unique peptides produced with an atmospheric pressure matrix‐assisted laser desorption/ionization source

“…Owing to the high degree of similarity between sequenced SASPs of Bacillus globigii and Bacillus stearothermophilus, 25 only a limited number of species-unique peptides were identified for each of these (Table 2). For Bacillus subtilis, whose genome is completely sequenced and whose SASPs differ in sequence to a larger extent from those of other species, numerous specific SASP peptides were identified for subsequent targeting.…”

“…SASP sequences for Bacillus globigii were sequenced in our laboratory. 25 In silico trypsin digestions were performed using PeptideCutter (http://us.expasy.org). Peptides that were found uniquely in a given Bacillus species were tabulated, and the species uniqueness of each was tested by performing a MASCOT sequence query (http://www.matrixscience.com) (see below for details of the search parameters).…”

“…5,24 In addition, it has recently been determined that the major SASPs of Bacillus globigii and Bacillus stearothermophilus are nearly identical. 25 In order to achieve more rapid, automatable species differentiation, it is advantageous to focus directly on species-unique peptide sequences. To expedite species characterization further, the overall analysis time could also be reduced by employing an instrument in which it is not necessary to bring the sample under vacuum.…”

Enhanced specificity of bacterial spore identification by oxidation and mass spectrometry

“…[1][2][3][4][5][6][7] The extreme resilience of bacterial endospores, 8 appropriate for their postulated role in universal panspermia, presents additional challenges for their rapid detection in the environment. A number of recent studies discuss applications of MALDI-MS for characterization of bacterial [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] or fungal [23][24][25][26] spores.…”