Dataset Information

Proteomic analysis of the thermophilic methylotroph Bacillus methanolicus MGA3: 50°C versus 37°C

ABSTRACT: Here, we established for the first time a proteomic analysis of B. methanolicus MGA3, and we used this approach to compare cells grown on methanol and mannitol at 50°C as well as cells grown on methanol at 37°C and 50°C. Bacillus methanolicus MGA3 is a facultative methylotroph of industrial relevance that is able to grow on methanol as its sole source of carbon and energy. The Gram-positive bacterium possesses a soluble NAD-dependent methanol dehydrogenase and assimilates formaldehyde via the ribulose monophosphate (RuMP) cycle. We used label-free quantitative proteomics to generate reference proteome data for this bacterium and compared the proteome of B. methanolicus MGA3 on two different carbon sources (methanol and mannitol) as well as two different growth temperatures (50°C and 37°C). From a total of approximately 1,200 different detected proteins, approximately 1,000 of these were used for quantification. While the levels of 213 proteins were significantly different at the two growth temperatures tested, the levels of 109 proteins changed significantly when cells were grown on different carbon sources. The carbon source strongly affected the expression of enzymes related to carbon metabolism, and in particular, both dissimilatory and assimilatory RuMP cycle enzyme levels were elevated during growth on methanol compared to mannitol. Our data also indicate that B. methanolicus has a functional TCA cycle that is differentially regulated on mannitol and methanol. Other proteins presumed to be involved in growth on methanol were constitutively expressed under the different growth conditions. Protein Identification: Raw data of gel slices with equal molecular weight were loaded together into the software package Progenesis LCMS Version. 4.1 (www.nonlinear.com), a software tool developed for label-free quantification of LC–MS data. Data was analyzed according to Progenesis LCMS analysis in Weisser et al., In brief, for data loading, the option "High Mass Accuracy Instrument" was selected. LC–MS data were normalized and aligned according to manufacturer’s specifications. In the aligning step manual seeding of "three to five" vectors along the retention time gradient was performed followed by automatic alignment. For the identification of peptide features in the corresponding mass spectrometry files, Mascot generic files (.mgf file format) are generated with Progenesis LCMS (using up to five tandem mass spectra for each feature with the top 200 fragment ion peaks ,charge deconvolution and de-isotopting activated). Mgf files were searched by Mascot 2.3 search engine against an amino acid database containing 20,399 entries including 3,418 MGA3 annotated proteins (downloaded from Uniprot on August 2013), 6,651 yeast proteins, 261 known mass spectrometry contaminants as forward entries and from all forward entries except the contaminats, a reverse decoy sequence. Parameters for precursor tolerance and fragment ion tolerance were set to ± 10 ppm and ± 0.6 Da, respectively. Mascot results were loaded into Scaffold v4.05, using the options for protein clustering and high mass accuracy PeptideProphet scoring.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Bacillus Methanolicus

SUBMITTER: Miriam Bortfeld-Miller

LAB HEAD: Miriam Bortfeld-Miller

PROVIDER: PXD000638 | Pride | 2014-01-28

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20130620_17_MeOH_R1_S7.RAW	Raw
	20130620_18_MeOH_R2_S7.RAW	Raw
	20130620_19_MeOH_R3_S7.RAW	Raw
	20130620_20_MeOH37_R1_S7.RAW	Raw
	20130620_21_MeOH37_R2_S7.RAW	Raw

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Proteomic analysis of the thermophilic methylotroph Bacillus methanolicus MGA3.

Müller Jonas E N JE Litsanov Boris B Bortfeld-Miller Miriam M Trachsel Christian C Grossmann Jonas J Brautaset Trygve T Vorholt Julia A JA

Proteomics 20140220 6

Bacillus methanolicus MGA3 is a facultative methylotroph of industrial relevance that is able to grow on methanol as its sole source of carbon and energy. The Gram-positive bacterium possesses a soluble NAD(+) -dependent methanol dehydrogenase and assimilates formaldehyde via the ribulose monophosphate (RuMP) cycle. We used label-free quantitative proteomics to generate reference proteome data for this bacterium and compared the proteome of B. methanolicus MGA3 on two different carbon sources (m ...[more]

PMID: 24452867

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Project description:Here, we established for the first time a proteomic analysis of B. methanolicus MGA3, and we used this approach to compare cells grown on methanol and mannitol at 50°C as well as cells grown on methanol at 37°C and 50°C. Bacillus methanolicus MGA3 is a facultative methylotroph of industrial relevance that is able to grow on methanol as its sole source of carbon and energy. The Gram-positive bacterium possesses a soluble NAD-dependent methanol dehydrogenase and assimilates formaldehyde via the ribulose monophosphate (RuMP) cycle. We used label-free quantitative proteomics to generate reference proteome data for this bacterium and compared the proteome of B. methanolicus MGA3 on two different carbon sources (methanol and mannitol) as well as two different growth temperatures (50°C and 37°C). From a total of approximately 1,200 different detected proteins, approximately 1,000 of these were used for quantification. While the levels of 213 proteins were significantly different at the two growth temperatures tested, the levels of 109 proteins changed significantly when cells were grown on different carbon sources. The carbon source strongly affected the expression of enzymes related to carbon metabolism, and in particular, both dissimilatory and assimilatory RuMP cycle enzyme levels were elevated during growth on methanol compared to mannitol. Our data also indicate that B. methanolicus has a functional TCA cycle that is differentially regulated on mannitol and methanol. Other proteins presumed to be involved in growth on methanol were constitutively expressed under the different growth conditions. Protein Identification: Raw data of gel slices with equal molecular weight were loaded together into the software package Progenesis LCMS Version. 4.1 (www.nonlinear.com), a software tool developed for label-free quantification of LC–MS data. Data was analyzed according to Progenesis LCMS analysis in Weisser et al., In brief, for data loading, the option "High Mass Accuracy Instrument" was selected. LC–MS data were normalized and aligned according to manufacturer’s specifications. In the aligning step manual seeding of "three to five" vectors along the retention time gradient was performed followed by automatic alignment. For the identification of peptide features in the corresponding mass spectrometry files, Mascot generic files (.mgf file format) are generated with Progenesis LCMS (using up to five tandem mass spectra for each feature with the top 200 fragment ion peaks ,charge deconvolution and de-isotopting activated). Mgf files were searched by Mascot 2.3 search engine against an amino acid database containing 20,399 entries including 3,418 MGA3 annotated proteins (downloaded from Uniprot on August 2013), 6,651 yeast proteins, 261 known mass spectrometry contaminants as forward entries and from all forward entries except the contaminats, a reverse decoy sequence. Parameters for precursor tolerance and fragment ion tolerance were set to ± 10 ppm and ± 0.6 Da, respectively. Mascot results were loaded into Scaffold v4.05, using the options for protein clustering and high mass accuracy PeptideProphet scoring.

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