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Differential Proteomics Based on Multidimensional Protein Identification Technology to Understand the Biogenesis of Outer Membrane of Escherichia coli

  • Alessandra M. Martorana
  • Sara Motta
  • Paola Sperandeo
  • Pierluigi MauriEmail author
  • Alessandra PolissiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1440)

Abstract

Cell envelope proteins in bacteria are typically difficult to characterize due to their low abundance, poor solubility, and the problematic isolation of pure surface fraction with only minimal contamination. Here we describe a method for cell membrane fractionation followed by mass spectrometry-based proteomics to analyze and determine protein abundance in bacterial membranes.

Key words

MudPIT Membrane proteins Gel-free Gram-negative cell envelope Outer membrane Cell fractionation 

Notes

Acknowledgements

We thank CNR Project “FaReBio di Qualità,” MIUR PRIN 2012WJSX8K, and Regione Lombardia Progetti di Ricerca Industriale e Sviluppo Sperimentale ID n. 30190679 for the financial support provided for this study. We thank Marta G. Bitonti for providing the MAProMA software.

References

  1. 1.
    Washburn MP, Wolters D, Yates JR 3rd (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 19:242–247CrossRefPubMedGoogle Scholar
  2. 2.
    Park SK, Venable JD, Xu T, Yates JR 3rd (2008) A quantitative analysis software tool for mass spectrometry-based proteomics. Nat Methods 5:319–322PubMedPubMedCentralGoogle Scholar
  3. 3.
    Mauri P, Scigelova M (2009) Multidimensional protein identification technology for clinical proteomic analysis. Clin Chem Lab Med 47:636–646CrossRefPubMedGoogle Scholar
  4. 4.
    De Palma A, Roveri A, Zaccarin M, Benazzi L, Daminelli S, Pantano G, Buttarello M, Ursini F, Gion M, Mauri PL (2010) Extraction methods of red blood cell membrane proteins for multidimensional protein identification technology (MudPIT) analysis. J Chromatogr A 1217:5328–5336CrossRefPubMedGoogle Scholar
  5. 5.
    Martorana AM, Motta S, Di Silvestre D, Falchi F, Dehò G, Mauri P, Sperandeo P, Polissi A (2014) Dissecting Escherichia coli outer membrane biogenesis using differential proteomics. PLoS One 9:e100941CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Polissi A, Sperandeo P (2014) The lipopolysaccharide export pathway in Escherichia coli: structure, organization and regulated assembly of the Lpt machinery. Mar Drugs 12:1023–1042CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Silhavy TJ, Kahne D, Walker S (2010) The bacterial cell envelope. Cold Spring Harb Perspect Biol 2:a000414CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Kinnebrew MA, Pamer EG (2011) Innate immune signaling in defense against intestinal microbes. Immunol Rev 245:113–131CrossRefGoogle Scholar
  9. 9.
    Thanassi DG, Bliska JB, Christie PJ (2012) Surface organelles assembled by secretion systems of Gram-negative bacteria: diversity in structure and function. FEMS Microbiol Rev 36:1046–1082CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Osborn MJ, Gander JE, Parisi E (1972) Mechanism of assembly of the outer membrane of Salmonella typhimurium. Site of synthesis of lipopolysaccharide. J Biol Chem 247:3973–3986PubMedGoogle Scholar
  11. 11.
    Sperandeo P, Cescutti R, Villa R, Di Benedetto C, Candia D, Dehò G, Polissi A (2007) Characterization of lptA and lptB, two essential genes implicated in lipopolysaccharide transport to the outer membrane of Escherichia coli. J Bacteriol 189:244–253CrossRefPubMedGoogle Scholar
  12. 12.
    Mauri P, Dehò G (2008) A proteomic approach to the analysis of RNA degradosome composition in Escherichia coli. Methods Enzymol 447:99–117CrossRefPubMedGoogle Scholar
  13. 13.
    Di Silvestre D, Brunetti P, Vella D, Brambilla F, De Palma A, Mauri P (2015) Automated extraction of proteotypic peptides by shotgun proteomic experiments: a new computational tool and two actual cases. Curr Biotechnol 4:39–45CrossRefGoogle Scholar
  14. 14.
    Mauri P, Scarpa A, Nascimbeni AC, Benazzi L, Parmagnani E, Mafficini A, Della Peruta M, Bassi C, Miyazaki K, Sorio C (2005) Identification of proteins released by pancreatic cancer cells by multidimensional protein identification technology: a strategy for identification of novel cancer markers. FASEB J 19:1125–1127PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Dipartimento di Biotecnologie e BioscienzeUniversità di Milano-BicoccaMilanItaly
  2. 2.Istituto di Tecnologie Biomediche, Consiglio Nazionale delle RicercheMilanItaly

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