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Comparative and Functional Genomics
Volume 3 (2002), Issue 6, Pages 470-483
http://dx.doi.org/10.1002/cfg.211
Research Article

Proteome Analysis of the Plasma Membrane of Mycobacterium tuberculosis

1Division of Biochemistry, Central Drug Research Institute, Chattar Manzil, Post Box No. 173, Lucknow 226001, India
2PT3 — Protéomique Génopole, Institut Pasteur, 28 Rue du Docteur Roux, Paris Cedex 15 75724, France
3Unité de Génétique Moléculaire Bactérienne, Institut Pasteur, 28 Rue du Docteur Roux, Paris Cedex 15 75724, France

Received 6 June 2002; Accepted 11 September 2002

Copyright © 2002 Hindawi Publishing Corporation. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The plasma membrane of Mycobacterium tuberculosis is likely to contain proteins that could serve as novel drug targets, diagnostic probes or even components of a vaccine against tuberculosis. With this in mind, we have undertaken proteome analysis of the membrane of M. tuberculosis H37Rv. Isolated membrane vesicles were extracted with either a detergent (Triton X114) or an alkaline buffer (carbonate) following two of the protocols recommended for membrane protein enrichment. Proteins were resolved by 2D-GE using immobilized pH gradient (IPG) strips, and identified by peptide mass mapping utilizing the M. tuberculosis genome database. The two extraction procedures yielded patterns with minimal overlap. Only two proteins, both HSPs, showed a common presence. MALDI–MS analysis of 61 spots led to the identification of 32 proteins, 17 of which were new to the M. tuberculosis proteome database. We classified 19 of the identified proteins as ‘membrane-associated’; 14 of these were further classified as ‘membrane-bound’, three of which were lipoproteins. The remaining proteins included four heat-shock proteins and several enzymes involved in energy or lipid metabolism. Extraction with Triton X114 was found to be more effective than carbonate for detecting ‘putative’ M. tuberculosis membrane proteins. The protocol was also found to be suitable for comparing BCG and M. tuberculosis membranes, identifying ESAT-6 as being expressed selectively in M. tuberculosis. While this study demonstrates for the first time some of the membrane proteins of M. tuberculosis, it also underscores the problems associated with proteomic analysis of a complex membrane such as that of a mycobacterium.