Elsevier

Journal of Microbiological Methods

Volume 131, December 2016, Pages 144-147
Journal of Microbiological Methods

Optimizing of a protein extraction method for Mycobacterium tuberculosis proteome analysis using mass spectrometry

https://doi.org/10.1016/j.mimet.2016.10.021Get rights and content

Highlights

  • Optimized protein extraction method for Mycobacterium tuberculosis is proposed.

  • Optimal extraction method combines urea an thiourea with sonication and bead beating.

  • Concentration of proteins may vary depending on the proportion of bead/buffer used.

Abstract

A critical step in proteomic analyses comprises the implementation of a reliable cell lysis method with high yields of qualitative proteins. In Mycobacteria, the protein extraction step is often hampered by the thick waxy cell wall which is rich in mycolic acids. Harsh disruption techniques to release proteins from the cells are thus required. Here, we demonstrate an optimized protein extraction procedure for Mycobacterium tuberculosis (Mbt) that results in protein extracts that are useful for all currently used proteomics platforms, including gel and LC-MS based strategies. We compared the effectiveness of using both thiourea and urea and/or SDS and DTT in the solubilization buffer, in combination or not with sonication and/or bead beating. After some preliminary optimization steps on fast-growing Mbt-like organisms, namely Mycobacterium smegmatis and Mycobacterium fortuitum, the final protein extraction protocol was tested on M. tuberculosis. Based on the concentrations of the proteins recovered from each of the tested methods and on the quality of the extracted proteins as evaluated by SDS PAGE, we propose a lysis buffer that contains both thiourea and urea, in combination with two mechanical cell disruption methods: sonication and bead beating. The optimized protocol results in protein extracts that are useful in M. tuberculosis proteomics studies based on any proteomics strategy or platform.

Introduction

Tuberculosis (TB) remains a global health problem worldwide, in particular in developing countries. Annually, there are an estimated 9.6 million new TB cases and 1.5 million TB deaths (WHO report, 2015). In addition, the TB situation is aggravated by the emergence of drug resistant strains, multidrug resistant (MDR) and extensively drug resistant (XDR).

As alternative to molecular detection of drug resistance, proteomics analysis, based on mass spectral analyses, has become recognized in systems biology studies and as an emerging tool for rapidly assessing drug resistance. New high-throughput platforms based on liquid chromatography hyphenated with tandem mass spectrometry (LC-MS/MS) have been developed and improved the accuracy of protein identification as their quantification, opening new ways for TB drug resistance studies (Silva et al., 2006, de Souza et al., 2010, Gillet et al., 2012).

A unique characteristic of mycobacteria is their thick waxy outer cell wall, consisting of mycolic acids and arabinogalactans associated with the peptidoglycan envelope (Brennan, 1995). Mycolic acids are extremely hydrophobic molecules that affect the permeability at the cell surface. The cell envelope helps mycobacteria to survive in extreme environmental conditions and the presence of antibiotics, thus playing an important role in drug resistance. The lipid shell around the organism renders mycobacteria highly resistant to common lysis techniques and, consequently, harsh disruption methods are required to release proteins from these cells. As such, proteomics analysis is often hampered by the complexity of the mycobacterial cell wall: thick, waxy, hydrophobic and rich in mycolic acids, requiring an optimized sample preparation. Sample preparation is a crucial step in proteomics studies, especially in comparative proteomics, where minor differences between experimental and control samples are searched. Currently, few protocols for protein extraction and solubilization of mycobacterial proteomes are available. Cell lysis of mycobacteria remains challenging: several cell lysis techniques including chemicals, sonication, French press disruption, bead beating and combinations thereof, have been employed in mycobacterial genomics, immunological and proteomics studies (Monahan et al., 2001, Betts et al., 2000, Odumeru et al., 2001, Hunter et al., 1990, Hurley et al., 1987, Mutharia et al., 1997, Jungblut et al., 1999, Rosenkrands et al., 2000a, Rosenkrands et al., 2000b, Lee et al., 1992, Yoshimura et al., 1987, Rabilloud et al., 1997, Herbert, 1999, Gorg et al., 2004). In this study, we compared and optimized different lysis buffers in combination with sonication and bead beating to extract the proteomes of several Mycobacterium species as a model for M. tuberculosis.

Section snippets

Methods

  • 2.1.

    Mycobacterial strains, growth and cell extraction

For safety and convenience reasons, fast growing mycobacteria under Biosafety Level 2 (BSL-2) Mycobacterium smegmatis CCUG 28063 and M. fortuitum CCUG 31556, obtained from the CCUG collection (http://www.ccug.se), were initially used. After optimization of the protein extraction method, the optimized protocol was used with the H37Rv M. tuberculosis strain. Bacteria were grown in 40 mL Middlebrook 7H9 liquid culture (BD Difco™ Middlebrook 7H9

Results

In order to optimize the sample preparation method for M. tuberculosis prior to proteomics analysis, we assessed four lysis buffers in combination with sonication and bead beating. Preliminary experiments on M. smegmatis and M fortuitum showed that adding both urea and thiourea in the lysis buffer resulted in higher protein extraction yields (Table 2). Whereas the addition of SDS resulted in similar yields as adding (thio)urea, the DTT-buffer did not result in higher protein yields as compared

Discussion

We compared different protein extraction methods including lysis buffer, sonication, and bead beating to set up an optimized method of protein extraction for subsequent proteomic analysis. The results show that, the optimal lysis buffer, independent of the mechanical cell wall disruption method used, contained both thiourea and urea Such buffers were originally designed by O'Farrell (1975) and it is commonly used as lysis buffer to improve the solubilization of proteins (Rabilloud, 1998). The

Conclusion

M. tuberculosis is known for its complex cell wall structure, which affects its membrane permeability and plays an important role in drug resistance. As the output of a proteomics experiment depends on the quality of the proteome extract, reliable methods for the quantitative extraction of intact proteins are of critical importance. In this study, a comparative analysis of different lysis methods was carried out for the extraction of proteins from mycobacteria. We demonstrated that a protein

Acknowledgements

MSR was a Fellow of «Les Amis des Instituts Pasteur de Bruxelles/Gent University» and is a Fellow of Pasteur Institute of Madagascar.

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