GeLC-MS-based proteomics of Chromobacterium violaceum: comparison of proteome changes elicited by hydrogen peroxide.

Chromobacterium violaceum is a free-living bacillus with several genes that enables it survival under different harsh environments such as oxidative and temperature stresses. Here we performed a label-free quantitative proteomic study to unravel the molecular mechanisms that enable C. violaceum to survive oxidative stress. To achieve this, total proteins extracted from control and C. violaceum cultures exposed during two hours with 8 mM hydrogen peroxide were analyzed using GeLC-MS proteomics. Analysis revealed that under the stress condition, the bacterium expressed proteins that protected it from the damage caused by reactive oxygen condition and decreasing the abundance of proteins responsible for bacterial growth and catabolism. GeLC-MS proteomics analysis provided an overview of the metabolic pathways involved in the response of C. violaceum to oxidative stress ultimately aggregating knowledge of the response of this organism to environmental stress. This study identified approximately 1500 proteins, generating the largest proteomic coverage of C. violaceum so far. We also detected proteins with unknown function that we hypothesize to be part of new mechanisms related to oxidative stress defense. Finally, we identified the mechanism of clustered regularly interspaced short palindromic repeats (CRISPR), which has not yet been reported for this organism.

Analysis of the salivary proteome in gingivitis patients.

BACKGROUND AND OBJECTIVE: Gingivitis is a disease that is characterized by inflammation of the gingival tissue, which can progress to periodontitis and tooth loss. Although many studies have attempted to identify salivary proteins that are associated with the disease, this is the first study to use a proteomic approach to analyze and compare the proteomic profile of whole saliva from gingivitis patients and healthy controls. MATERIAL AND METHOD: To analyze the saliva proteome, two-dimensional gel electrophoresis and liquid chromatography were used, followed by mass spectrometry. RESULTS: The analyses showed that gingival inflammation was associated with increased amounts of blood proteins (serum albumin and hemoglobin), immunoglobulin peptides and keratins. In the control group, salivary cystatins, which were detected using capillary Liquid Chromatography on line to electrospray ionization Quadrupole Time-of-flight mass spectrometry, appeared to be more abundant. CONCLUSION: This approach provides novel insight into profiles of the salivary proteome during gingival inflammation, which may contribute to improvements in diagnosis.

Proteome analysis of embryogenic cell suspensions of cowpea (Vigna unguiculata).

Using a combination of two-dimensional gel electrophoresis protein mapping and mass spectrometry analysis, we have established proteome reference maps of embryogenic cell suspensions of cowpea (Vigna unguiculata). The cell suspensions were generated from young primary leaves and contained basically pro-embryogenic masses, which enabled us to dissect their proteome composition while eliminating the complexity of too many cell types. Over 550 proteins could reproducibly be resolved over a pI range of 3-10. A total of 128 of the most abundant protein spots were excised, digested in-gel with trypsin and analyzed by tandem mass spectrometry. This enabled the identification of 67 protein spots. Two of the most abundant proteins were identified as a chitinase and as a ribonuclease belonging to the family of PR-4 and PR-10 proteins, respectively. The expression of the respective genes was confirmed by RT-PCR and the pattern of deposition of the PR-10 protein in cell suspensions as well as in developing cowpea seeds, roots, shoots and flowers were determined by Western blot experiments, using synthetic antibodies raised against a 14-amino acid synthetic peptide located close to the C-terminal region of the PR-10 protein.