Bacillus cereus strains fall into two clusters (one closely and one more distantly related) to Bacillus anthracis according to amino acid substitutions in small acid-soluble proteins as determined by tandem mass spectrometry.

Small acid-soluble proteins (SASPs) are located in the core region of Bacillus spores and have been previously demonstrated as reliable biomarkers for differentiating Bacillus anthracis and Bacillus cereus. Using MS and MS-MS analysis of SASPs further phylogenetic correlations among B. anthracis and B. cereus strains are described here. ESI was demonstrated to be a more comprehensive method, allowing for the analysis of intact proteins in both MS and MS-MS mode, thus providing molecular weight (MW) and sequence information in a single analysis, and requiring almost no sample preparation. MALDI MS was used for determination of MW of intact proteins; however, MS-MS analysis can only be achieved after enzymatic digestion of these proteins. It was demonstrated that the combination of the two different approaches provides confirmatory and complementary information, allowing for unambiguous protein characterization and sequencing. This study established that B. cereus strains fall into two clusters (one closely and one more distantly related) to B. anthracis as exhibited by amino acid substitutions. The closely related cluster was characterized by a beta-SASP with a single amino acid substitution, localized either close to the C terminus (phenylalanine-->tyrosine, 16 masses change) or close to the N terminus (serine-->alanine serine, also 16 masses change). The more distantly related cluster displayed both amino acid substitutions (32 masses change). One strain of B. cereus isolated from a patient with severe pneumonia (an anthrax-like disease) fell into the more distantly related cluster implying that pathogenicity and phylogenicity are not necessarily correlated features. Unlike PCR and DNA sequencing, protein sequence variation assessed by ESI MS-MS, essentially occurs in real-time, and involves simply extracting the protein and injecting into the instrument for analysis.

Proteome annotations and identifications of the human pulmonary fibroblast.

We hereby report on a three year project initiative undertaken by our research team encompassing large-scale protein expression profiling and annotations of human primary lung fibroblast cells. An overview is given of proteomic studies of the fibroblast target cell involved in several diseases such as asthma, idiopatic pulmonary disease, and COPD. It has been the objective within our research team to map and identify the protein expressions occurring in both activated-, as well as resting cell states. The JGGL database www.2DDB.org has been built around these data, allowing advanced hypothesis building using the interactive query bioinformatic tools developed. Gene ontology has been applied to these annotations, classifying and correlating protein expressions to function. The localization as well as the biological processes involved for the annotations are being presented including an annotation-, and sequence-identification strategy, resulting in close to 2000 protein identities. Both gel based, high resolution 2D-gels, and liquid-phase separation (three-dimensional HPLC), as well as the combination of gel- and LC-based approaches (1D-gels and nano-capillary LC, reversed-phase) were utilized. Protein sequencing and structure identities were acquired by a combination of MALDI-, and electrospray-mass spectrometry techniques. Phenotypical and morphological characterizations were also made for this human disease target cell in both stimulated- and resting-cell states. The use of functional assays that demonstrate the key regulating role of growth factors and cytokine stimuli such as PDGF, TGF-beta, and EGF and the effect of ECM molecules such as Biglycan, are also presented and discussed.