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Definition of novel cell envelope associated proteins in Triton X-114 extracts of Mycobacterium tuberculosis H37Rv.

Målen H, Pathak S, Søfteland T, de Souza GA, Wiker HG - BMC Microbiol. (2010)

Bottom Line: Also, 64 of the identified proteins were predicted lipoproteins, and 54 proteins were predicted as outer membrane proteins.Three-hundred-and-ninety-five of the observed proteins, including 91 integral membrane proteins were described for the first time.The outcome showed that among the membrane-and membrane-associated proteins several proteins are present with high relative abundance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Section for Microbiology and Immunology, the Gade Institute, University of Bergen, Bergen, Norway.

ABSTRACT

Background: Membrane- and membrane-associated proteins are important for the pathogenicity of bacteria. We have analysed the content of these proteins in virulent Mycobacterium tuberculosis H37Rv using Triton X-114 detergent-phase separation for extraction of lipophilic proteins, followed by their identification with high resolution mass spectrometry.

Results: In total, 1417 different proteins were identified. In silico analysis of the identified proteins revealed that 248 proteins had at least one predicted trans-membrane region. Also, 64 of the identified proteins were predicted lipoproteins, and 54 proteins were predicted as outer membrane proteins. Three-hundred-and-ninety-five of the observed proteins, including 91 integral membrane proteins were described for the first time. Comparison of abundance levels of the identified proteins was performed using the exponentially modified protein abundance index (emPAI) which takes into account the number of the observable peptides to the number of experimentally observed peptide ions for a given protein. The outcome showed that among the membrane-and membrane-associated proteins several proteins are present with high relative abundance. Further, a close examination of the lipoprotein LpqG (Rv3623) which is only detected in the membrane fractions of M. tuberculosis but not in M. bovis, revealed that the homologous gene in M. bovis lack the signal peptide and lipobox motif, suggesting impaired export to the membrane.

Conclusions: Altogether, we have identified a substantial proportion of membrane- and membrane-associated proteins of M. tuberculosis H37Rv, compared the relative abundance of the identified proteins and also revealed subtle differences between the different members of the M. tuberculosis complex.

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SDS-PAGE analysis of the extracted M. tuberculosis H37Rv proteins. Panel A, shows the whole cell lysate of M. tuberculosis H37Rv, the aqueous phase proteins and the lipid phase proteins after Triton X-114 extraction. The fractions for LC-MS/MS analysis of the lipid phase is indicated. Explanation of the fraction numbers: (1) >160 kDa, (2) 105-160 kDa, (3) 75-105 kDa, (4) 50-75 kDa, (5) 35-50 kDa, (6) 30-35 kDa, (7) 25-30 kDa, (8) 15-25 kDa, (9) 15-10 kDa, (10) <10 kDa. Panel B shows western blot analysis of the aqueous and lipid phases using a polyclonal rabbit antiserum against a BCG cell wall fraction. The molecular weight standards are shown on the left hand side of each panel.
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Figure 1: SDS-PAGE analysis of the extracted M. tuberculosis H37Rv proteins. Panel A, shows the whole cell lysate of M. tuberculosis H37Rv, the aqueous phase proteins and the lipid phase proteins after Triton X-114 extraction. The fractions for LC-MS/MS analysis of the lipid phase is indicated. Explanation of the fraction numbers: (1) >160 kDa, (2) 105-160 kDa, (3) 75-105 kDa, (4) 50-75 kDa, (5) 35-50 kDa, (6) 30-35 kDa, (7) 25-30 kDa, (8) 15-25 kDa, (9) 15-10 kDa, (10) <10 kDa. Panel B shows western blot analysis of the aqueous and lipid phases using a polyclonal rabbit antiserum against a BCG cell wall fraction. The molecular weight standards are shown on the left hand side of each panel.

Mentions: The aim of this study was to enrich and perform a comprehensive proteomic analysis of membrane- and membrane-associated proteins of the virulent reference strain M. tuberculosis H37Rv. For this purpose, the hydrophobic proteins were enriched by lysing whole bacilli followed by phase separation with the Triton X-114 detergent. After phase separation, the proteins in the lipid phase were precipitated by acetone and separated by SDS-PAGE. As shown in Figure 1 panel A, the lipid phase was quite complex, but appeared to be enriched for certain proteins as compared to the unfractionated crude lysate. In a parallel experiment, and to validate that the protein content in the lipid and aqueous phases were different, proteins from both phases were separated and transferred to nitrocellulose membranes which were developed with polyclonal antibodies against a cell wall fraction of M. bovis BCG (Figure 1, panel B). Notably, Figure 1 not only demonstrates that the protein content of the aqueous phase and the lipid phase was different, but also clearly shows that the lipid phase was indeed enriched for cell wall proteins. In order to identify the proteins of the Triton X-114 detergent fraction, the protein mixture was separated with SDS-PAGE (Figure 1A), run in duplicate and cut into ten pieces each (twenty fractions in total) and subjected to in-gel digestion by trypsin. The resulting peptides were eluted and analysed by high accuracy mass spectrometry. Additional file 1, Figure S1 illustrates the sequence obtained for ion m/z 1210.62 which was identified by Mascot as peptide CGSPAWDLPTVFGPIAITYNIK from protein Rv0932c with a Mascot score of 79. Such fragmentation data contain a very good coverage of the expected y- and b-series daughter ions plus the presence of other ions which indicates the correct MS/MS assignment such as two highly abundant y-ions of proline (y19++ and y14). This is very typical for peptides containing proline.


Definition of novel cell envelope associated proteins in Triton X-114 extracts of Mycobacterium tuberculosis H37Rv.

Målen H, Pathak S, Søfteland T, de Souza GA, Wiker HG - BMC Microbiol. (2010)

SDS-PAGE analysis of the extracted M. tuberculosis H37Rv proteins. Panel A, shows the whole cell lysate of M. tuberculosis H37Rv, the aqueous phase proteins and the lipid phase proteins after Triton X-114 extraction. The fractions for LC-MS/MS analysis of the lipid phase is indicated. Explanation of the fraction numbers: (1) >160 kDa, (2) 105-160 kDa, (3) 75-105 kDa, (4) 50-75 kDa, (5) 35-50 kDa, (6) 30-35 kDa, (7) 25-30 kDa, (8) 15-25 kDa, (9) 15-10 kDa, (10) <10 kDa. Panel B shows western blot analysis of the aqueous and lipid phases using a polyclonal rabbit antiserum against a BCG cell wall fraction. The molecular weight standards are shown on the left hand side of each panel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2874799&req=5

Figure 1: SDS-PAGE analysis of the extracted M. tuberculosis H37Rv proteins. Panel A, shows the whole cell lysate of M. tuberculosis H37Rv, the aqueous phase proteins and the lipid phase proteins after Triton X-114 extraction. The fractions for LC-MS/MS analysis of the lipid phase is indicated. Explanation of the fraction numbers: (1) >160 kDa, (2) 105-160 kDa, (3) 75-105 kDa, (4) 50-75 kDa, (5) 35-50 kDa, (6) 30-35 kDa, (7) 25-30 kDa, (8) 15-25 kDa, (9) 15-10 kDa, (10) <10 kDa. Panel B shows western blot analysis of the aqueous and lipid phases using a polyclonal rabbit antiserum against a BCG cell wall fraction. The molecular weight standards are shown on the left hand side of each panel.
Mentions: The aim of this study was to enrich and perform a comprehensive proteomic analysis of membrane- and membrane-associated proteins of the virulent reference strain M. tuberculosis H37Rv. For this purpose, the hydrophobic proteins were enriched by lysing whole bacilli followed by phase separation with the Triton X-114 detergent. After phase separation, the proteins in the lipid phase were precipitated by acetone and separated by SDS-PAGE. As shown in Figure 1 panel A, the lipid phase was quite complex, but appeared to be enriched for certain proteins as compared to the unfractionated crude lysate. In a parallel experiment, and to validate that the protein content in the lipid and aqueous phases were different, proteins from both phases were separated and transferred to nitrocellulose membranes which were developed with polyclonal antibodies against a cell wall fraction of M. bovis BCG (Figure 1, panel B). Notably, Figure 1 not only demonstrates that the protein content of the aqueous phase and the lipid phase was different, but also clearly shows that the lipid phase was indeed enriched for cell wall proteins. In order to identify the proteins of the Triton X-114 detergent fraction, the protein mixture was separated with SDS-PAGE (Figure 1A), run in duplicate and cut into ten pieces each (twenty fractions in total) and subjected to in-gel digestion by trypsin. The resulting peptides were eluted and analysed by high accuracy mass spectrometry. Additional file 1, Figure S1 illustrates the sequence obtained for ion m/z 1210.62 which was identified by Mascot as peptide CGSPAWDLPTVFGPIAITYNIK from protein Rv0932c with a Mascot score of 79. Such fragmentation data contain a very good coverage of the expected y- and b-series daughter ions plus the presence of other ions which indicates the correct MS/MS assignment such as two highly abundant y-ions of proline (y19++ and y14). This is very typical for peptides containing proline.

Bottom Line: Also, 64 of the identified proteins were predicted lipoproteins, and 54 proteins were predicted as outer membrane proteins.Three-hundred-and-ninety-five of the observed proteins, including 91 integral membrane proteins were described for the first time.The outcome showed that among the membrane-and membrane-associated proteins several proteins are present with high relative abundance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Section for Microbiology and Immunology, the Gade Institute, University of Bergen, Bergen, Norway.

ABSTRACT

Background: Membrane- and membrane-associated proteins are important for the pathogenicity of bacteria. We have analysed the content of these proteins in virulent Mycobacterium tuberculosis H37Rv using Triton X-114 detergent-phase separation for extraction of lipophilic proteins, followed by their identification with high resolution mass spectrometry.

Results: In total, 1417 different proteins were identified. In silico analysis of the identified proteins revealed that 248 proteins had at least one predicted trans-membrane region. Also, 64 of the identified proteins were predicted lipoproteins, and 54 proteins were predicted as outer membrane proteins. Three-hundred-and-ninety-five of the observed proteins, including 91 integral membrane proteins were described for the first time. Comparison of abundance levels of the identified proteins was performed using the exponentially modified protein abundance index (emPAI) which takes into account the number of the observable peptides to the number of experimentally observed peptide ions for a given protein. The outcome showed that among the membrane-and membrane-associated proteins several proteins are present with high relative abundance. Further, a close examination of the lipoprotein LpqG (Rv3623) which is only detected in the membrane fractions of M. tuberculosis but not in M. bovis, revealed that the homologous gene in M. bovis lack the signal peptide and lipobox motif, suggesting impaired export to the membrane.

Conclusions: Altogether, we have identified a substantial proportion of membrane- and membrane-associated proteins of M. tuberculosis H37Rv, compared the relative abundance of the identified proteins and also revealed subtle differences between the different members of the M. tuberculosis complex.

Show MeSH
Related in: MedlinePlus