Advertisement

Proteomics of Vibrio cholerae

  • Ryszard A. ZielkeEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1839)

Abstract

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 words

Proteomics iTRAQ Secreted proteins Cell envelope 2D-LC-MS/MS 

References

  1. 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
  2. 2.
    Doro F et al (2009) Surfome analysis as a fast track to vaccine discovery: identification of a novel protective antigen for Group B Streptococcus hypervirulent strain COH1. Mol Cell Proteomics 8:1728–1737.  https://doi.org/10.1074/mcp.M800486-MCP200 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Rodriguez-Ortega MJ et al (2006) Characterization and identification of vaccine candidate proteins through analysis of the group A Streptococcus surface proteome. Nat Biotechnol 24:191–197.  https://doi.org/10.1038/nbt1179 CrossRefPubMedGoogle Scholar
  4. 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. 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
  6. 6.
    Ross PL et al (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3:1154–1169.  https://doi.org/10.1074/mcp.M400129-MCP200 CrossRefPubMedGoogle Scholar
  7. 7.
    Chahrour O, Cobice D, Malone J (2015) Stable isotope labelling methods in mass spectrometry-based quantitative proteomics. J Pharm Biomed Anal 113:2–20.  https://doi.org/10.1016/j.jpba.2015.04.013 CrossRefPubMedGoogle Scholar
  8. 8.
    Rauniyar N, Yates JR 3rd (2014) Isobaric labeling-based relative quantification in shotgun proteomics. J Proteome Res 13:5293–5309.  https://doi.org/10.1021/pr500880b CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Pharmaceutical Sciences, College of PharmacyOregon State UniversityCorvallisUSA

Personalised recommendations