Proteomics of Vibrio cholerae
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Combining high-throughput mass spectrometry with isobaric tags for relative and absolute quantification (iTRAQ) allows for the identification and relative quantification of proteins from multiple samples. Furthermore, low-abundance proteins that are usually not detected can be enriched by using only the relevant fraction of the proteome, e.g., cytoplasmic, membrane proteins, or secreted proteins. Described here is a workflow for isolation and enrichment of secreted and membrane proteins that is compatible with mass spectrometry. Isolated proteins are reduced, alkylated, and digested with trypsin, and obtained peptides are labeled with iTRAQ reagent and separated by strong cation exchange to reduce the complexity. Finally, the peptides are separated by reverse-phase chromatography, spotted on a MALDI target plate, and analyzed by MALDI TOF-TOF.
Key wordsProteomics iTRAQ Secreted proteins Cell envelope 2D-LC-MS/MS
- 1.Sikora AE, Zielke RA, Lawrence DA, Andrews PC, Sandkvist M (2011) Proteomic analysis of the Vibrio cholerae type II secretome reveals new proteins, including three related serine proteases. J Biol Chem 286:16555–16566. https://doi.org/10.1074/jbc.M110.211078 CrossRefPubMedPubMedCentralGoogle Scholar
- 4.Zielke RA et al (2016) Proteomics-driven antigen discovery for development of vaccines against gonorrhea. Mol Cell Proteomics. https://doi.org/10.1074/mcp.M116.058800
- 5.Zielke RA, Wierzbicki IH, Weber JV, Gafken PR, Sikora AE (2014) Quantitative proteomics of the Neisseria gonorrhoeae cell envelope and membrane vesicles for the discovery of potential therapeutic targets. Mol Cell Proteomics 13:1299–1317. https://doi.org/10.1074/mcp.M113.029538 CrossRefPubMedPubMedCentralGoogle Scholar