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Two-Dimensional Gel Electrophoresis-Based Proteomics of Mycobacteria

  • Jens Mattow
  • Frank Siejak
  • Kristine Hagens
  • Julia Kreuzeder
  • Stefan H.E. Kaufmann
  • Ulrich E. Schaible
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 465)

Abstract

Two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry (MS) is the classic proteomics approach used to monitor the dynamics of protein abundance and posttranslational modifications in biological systems. In this chapter, we provide detailed protocols for 2-DE–based proteomics of mycobacteria. Adequate standard operating procedures for mycobacterial culture, subcellular fractionation, and selective enrichment of proteins are indispensable prerequisites for targeted proteome analyses. Therefore, we also provide approved protocols for selective and efficient extraction of cytosolic, secreted, and hydrophobic plasma membrane proteins of mycobacteria, as well as for isolation of mycobacteria from infected macrophages.

Keywords

mass spectrometry mycobacteria Mycobacterium tuberculosis proteomics tandem mass spectrometry two-dimensional electrophoresis 

Notes

Acknowledgments

The authors thank Dr. Peter Jungblut (MPIIB), Dr. Frank Schmidt (University of Oslo, Biotechnology Centre of Oslo, Oslo, Norway), and Dr. Achim Treumann (Royal College of Surgeons, Department of Clinical Pharmacology, Dublin, Ireland) for their fruitful cooperation, Dr. Sarah Fortune (Harvard School of Public Health, Department of Immunology and Infectious Diseases, Boston, MA) for providing us with the protocol described in Section 8.3.1.3, Dr. Stephen Reece (MPIIB) for critical reading of the manuscript, and the Bundesministerium für Bildung und Forschung (Competence Network “Neue Methoden zur Erfassung des Gesamtproteoms von Bakterien” J.M., S.H.E.K., U.E.S.; Competence network “PathoGenoMik-Plus”; J.M., S.H.E.K.), the Deutsche Forschungsgemeinschaft (DFG Priority Programme SPP1131; U.E.S.), the Royal Society Wolfson Research Merit Award, UK (U.E.S.), and the European Community (Project “Structural and Functional Genomics of M. tuberculosis”; S.H.E.K.) for financial support.

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Jens Mattow
    • 1
  • Frank Siejak
    • 2
  • Kristine Hagens
    • 2
  • Julia Kreuzeder
    • 2
  • Stefan H.E. Kaufmann
    • 2
  • Ulrich E. Schaible
    • 2
  1. 1.Department of ImmunologyMax Planck Institute for Infection BiologyBerlinGermany
  2. 2.Department of ImmunologyMax Planck Institute for Infection BiologyBerlinGermany

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