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

Rabodoarivelo, Marie Sylvianne; Aerts, Maarten; Vandamme, Peter; Palomino, Juan Carlos; Rasolofo, Voahangy; Martin, Anandi

doi:10.1016/j.mimet.2016.10.021

Cited by 21 publications

(15 citation statements)

References 27 publications

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“…Proteins in Mycobacterium tuberculosis could be analyzed via many methods, mass spectrometry, codon usage, and so on [ 25 , 26 ]. Codon usage is linked to nucleic acids and proteins, especially the GC content.…”

Section: Resultsmentioning

confidence: 99%

Comprehensive Analysis and Comparison on the Codon Usage Pattern of Whole Mycobacterium tuberculosis Coding Genome from Different Area

Ren

Haixian

et al. 2018

BioMed Research International

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Phenomenon of unequal use of synonymous codons in Mycobacterium tuberculosis is common. Codon usage bias not only plays an important regulatory role at the level of gene expression, but also helps in improving the accuracy and efficiency of translation. Meanwhile, codon usage pattern of Mycobacterium tuberculosis genome is important for interpreting evolutionary characteristics in species. In order to investigate the codon usage pattern of the Mycobacterium tuberculosis genome, 12 Mycobacterium tuberculosis genomes from different area are downloaded from the GeneBank. The correlations between G3, GC12, whole GC content, codon adaptation index, codon bias index, and so on of Mycobacterium tuberculosis genomes are calculated. The ENC-plot, relationship between A3/(A3 + T3) and G3/(G3 + C3), GC12 versus GC3 plot, and the RSCU of overall/separated genomes all show that the codon usage bias exists in all 12 Mycobacterium tuberculosis genomes. Lastly, relationship between CBI and the equalization of ENC shows a strong negative correlation between them. The relationship between protein length and GC content (GC3 and GC12) shows that more obvious differences in the GC content may be in shorter protein. These results show that codon usage bias existing in the Mycobacterium tuberculosis genomes could be used for further study on their evolutionary phenomenon.

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Section: Resultsmentioning

confidence: 99%

Comprehensive Analysis and Comparison on the Codon Usage Pattern of Whole Mycobacterium tuberculosis Coding Genome from Different Area

Ren

Haixian

et al. 2018

BioMed Research International

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“…The transformants were selected on LB-Kan-Cm agar plates, then grown at 37°C in LB supplemented with 34 μg/ml Cm and 0.05% Tween 80 (to prevent cell clumping) and harvested in exponential phase (OD 600 ∼0.7-0.8). Protein extracts were prepared as described (54) with slight modifications. Cell pellets were resuspended in PBS and broken by using Beat Beater and 0,1 mm zircon beads (BioSpec Products) (3 times for 30 s on ice, with 1:4 vol/vol ratio of beads to cell suspension).…”

Section: Methodsmentioning

confidence: 99%

Regulation of ribosomal protein synthesis in mycobacteria: autogenous control ofrpsO

Boni

Aseev

Koledinskaya

et al. 2021

Preprint

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Autogenous regulation of ribosomal protein (r-protein) synthesis plays a key role in maintaining the stoichiometry of ribosomal components in bacteria. Our main goal was to develop techniques for investigating the r-protein synthesis regulation in mycobacteria, Gram-positive organisms with a high GC-content, which has never been addressed. We started with the rpsO gene known to be autoregulated by its product, r-protein S15, in a broad range of bacterial species. To study the in vivo regulation of rpsO from Mycobacterium smegmatis (Msm), we first applied an approach based on chromosomally integrated Msm rpsO'-'lacZ reporters by using E. coli as a surrogate host. The β-galactosidase assay has shown that mycobacterial rpsO expression is feedback regulated at the translation level in the presence of Msm S15 in trans, like in E. coli. Next, to overcome difficulties caused by the inefficiency of mycobacterial gene expression in E. coli, we created a fluorescent reporter system based on M. smegmatis. To this end, the integrative shuttle plasmid pMV306 was modified to provide insertion of the Msm or Mtb (M. tuberculosis) rpsO-egfp reporters into the Msm chromosome, and a novel E. coli- mycobacteria replicative shuttle vector, pAMYC, a derivative of pACYC184, was built. Analysis of the eGFP expression in the presence of the pAMYC derivative expressing MsmrpsO vs an empty vector confirms the autogenous regulation of the rpsO gene in mycobacteria. Additionally, we have revealed that the mycobacterial rpsO core promoters are rather weak and require upstream activating elements to enhance their strength.

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“…Proteins were extracted using an optimized protocol for mass spectrometry follow-up. 58 Cells were collected, washed with ice-cold PBS, and resuspended in 1mL lysis buffer (50 mM NH 4 HCO 3 pH 7.4, 10 mM MgCl 2 , 0.1% NaN 3 , 1 mM EGTA, 1 x protease inhibitors (Roche), 7 M urea, and 2 M thiourea). Cells were lysed with zirconia beads and the cell lysate was collected and filtered through a 0.22 µm membrane.…”

Section: Methodsmentioning

confidence: 99%

Efficacy of epetraborole against Mycobacterium abscessus is increased with norvaline

Sullivan

Lupien

Kalthoff

et al. 2021

Preprint

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Certain aminoacyl-tRNA synthetases developed a proofreading mechanism to ensure aminoacylation of tRNAs with cognate amino acids. Epetraborole (EPT) was identified as an inhibitor of the leucyl-tRNA synthetase (LeuRS) editing site in Mycobacterium abscessus. EPT displayed enhanced activity against M. abscessus over Mycobacterium tuberculosis. Crystallographic and equilibrium binding data showed that EPT binds LeuRSMabs and LeuRSMtb with similar Kd. Proteomic analysis revealed that when M. abscessus LeuRS mutants were fed the non-proteinogenic amino acid norvaline, leucine residues in proteins were replaced by norvaline, inducing expression of GroEL chaperonins and Clp proteases. In vitro data revealed that supplementation of media with norvaline reduced the emergence of EPT mutants in both M. abscessus and M. tuberculosis. The combination of EPT and norvaline had improved in vivo efficacy compared to EPT in a murine model of M. abscessus infection.

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

Cited by 21 publications

References 27 publications

Comprehensive Analysis and Comparison on the Codon Usage Pattern of Whole Mycobacterium tuberculosis Coding Genome from Different Area

Comprehensive Analysis and Comparison on the Codon Usage Pattern of Whole Mycobacterium tuberculosis Coding Genome from Different Area

Regulation of ribosomal protein synthesis in mycobacteria: autogenous control ofrpsO

Efficacy of epetraborole against Mycobacterium abscessus is increased with norvaline

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