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Proteomics analyses of the opportunistic pathogen Burkholderia vietnamiensis using protein fractionations and mass spectrometry.

ABSTRACT: The main objectives of this work were to obtain a more extensive coverage of the Burkholderia vietnamiensis proteome than previously reported and to identify virulence factors using tandem mass spectrometry. The proteome of B. vietnamiensis was precipitated into four fractions to as extracellular, intracellular, cell surface and cell wall proteins. Two different approaches were used to analyze the proteins. The first was a gel-based method where 1D SDS-PAGE was used for separation of the proteins prior to reverse phase liquid chromatography tandem mass spectrometry (LC-MS/MS). The second method used MudPIT analysis (Multi dimensional Protein Identification Technique), where proteins are digested and separated using cation exchange and reversed phase separations before the MS/MS analysis (LC/LC-MS/MS). Overall, gel-based LC-MS/MS analysis resulted in more protein identifications than the MudPIT analysis. Combination of the results lead to identification of more than 1200 proteins, approximately 16% of the proteins coded from the annotated genome of Burkholderia species. Several virulence factors were detected including flagellin, porin, peroxiredoxin and zinc proteases.

SUBMITTER: Wickramasekara S

PROVIDER: S-EPMC3237022 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Proteomics analyses of the opportunistic pathogen Burkholderia vietnamiensis using protein fractionations and mass spectrometry.

Wickramasekara Samanthi S Neilson Julie J Patel Naren N Breci Linda L Hilderbrand Amy A Maier Raina M RM Wysocki Vicki V

Journal of biomedicine & biotechnology 20111201

The main objectives of this work were to obtain a more extensive coverage of the Burkholderia vietnamiensis proteome than previously reported and to identify virulence factors using tandem mass spectrometry. The proteome of B. vietnamiensis was precipitated into four fractions to as extracellular, intracellular, cell surface and cell wall proteins. Two different approaches were used to analyze the proteins. The first was a gel-based method where 1D SDS-PAGE was used for separation of the protein ...[more]

PMID: 22187530

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Project description:Canine oral tumors are relatively common neoplasms in dogs. For disease monitoring and early diagnosis, salivary biomarkers are appropriate because saliva collection is non-invasive and requires no professional skills. In the era of omics, matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF MS) coupled with liquid chromatography-tandem MS (LC-MS/MS) are suitable to identify potential disease-associated peptides and proteins. The present study aimed to use MALDI-TOF MS and LC-MS/MS to search for particular peptide mass fingerprints (PMFs) and conceivable biomarkers in saliva of dogs with early- and late-stage oral melanoma (EOM and LOM, respectively), oral squamous cell carcinoma (OSCC), benign oral tumors (BN), and periodontitis and healthy controls (CP). Pooled saliva samples in each group were used to be representative of population change. Unique PMFs were obtained and specific peptide fragments were sequenced by LC-MS/MS and BLAST-searched with mammalian protein databases. Seven peptide fragments appeared in the tumor groups (EOM, LOM, OSCC and BN) at 1096, 1208, 1322, 1794, 1864, 2354 and 2483 Da, two peptide fragments appeared in the LOM and OSCC groups at 2450 and 3492 Da, and in the CP controls at 2544 and 3026 Da. Also, protein-chemotherapy drug interaction networks were exhibited. Using western blot analysis, the expression of sentrin-specific protease 7 (SENP7), a peptide fragment at 1096 Da, in OSCC was significantly increased, as was the expression of TLR4, a peptide fragment at 3492 Da, in LOM and OSCC, compared with the CP group. The expression of nuclear factor kappa B (NF-κB), a TLR4 partner, was notably increased in OSCC compared with CP, BN and EOM. The expression was also enhanced in LOM compared with EOM. Expressed protein sequences from western blots were verified by LC-MS/MS. Western blots were then performed with individual samples in each group. The results showed the elevated expression of TLR4 in LOM and OSCC, compared with that in CP and BN, the increased expression of NF-κB in LOM and OSCC, compared with CP and in LOM compared with BN, and the enhanced expression of SENP7 in LOM and OSCC, compared with that in CP and BN. In conclusion, discrete clusters of EOM, LOM, OSCC, BN and CP groups and potential protein candidates associated with the diseases were demonstrated by salivary proteomics. Western blot analysis verified SENP7, TLR4 and NF-κB as potential salivary biomarkers of canine oral tumors.

Dataset Information

Proteomics analyses of the opportunistic pathogen Burkholderia vietnamiensis using protein fractionations and mass spectrometry.

Publications

Proteomics analyses of the opportunistic pathogen Burkholderia vietnamiensis using protein fractionations and mass spectrometry.

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