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in the Proteomics Era

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  • Authors: Martin Gengenbacher1, Jeppe Mouritsen2, Olga T. Schubert3, Ruedi Aebersold4, Stefan H. E. Kaufmann6
  • Editors: Graham F. Hatfull7, William R. Jacobs Jr.8
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Max Planck Institute for Infection Biology, Department of Immunology, Charitéplatz 1, 10117 Berlin, Germany; 2: Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Wolfgang-Pauli Strasse 16, 8093 Zurich, Switzerland; 3: Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Wolfgang-Pauli Strasse 16, 8093 Zurich, Switzerland; 4: Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Wolfgang-Pauli Strasse 16, 8093 Zurich, Switzerland; 5: Faculty of Science, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland; 6: Max Planck Institute for Infection Biology, Department of Immunology, Charitéplatz 1, 10117 Berlin, Germany; 7: University of Pittsburgh, Pittsburgh, PA; 8: Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, NY
    • Source: microbiolspec March 2014 vol. 2 no. 2 doi:10.1128/microbiolspec.MGM2-0020-2013
  • Received 11 June 2013 Accepted 26 July 2013 Published 21 March 2014
  • M. Gengenbacher, [email protected]; S. Kaufmann, [email protected]
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  • Abstract:

    The emerging field of proteomics has contributed greatly to improving our understanding of the human pathogen over the last two decades. In this chapter we provide a comprehensive overview of mycobacterial proteome research and highlight key findings. First, studies employing a combination of two-dimensional gel electrophoresis and mass spectrometry (MS) provided insights into the proteomic composition, initially of the whole bacillus and subsequently of subfractions, such as the cell wall, cytosol, and secreted proteins. Comparison of results obtained under various culture conditions, i.e., acidic pH, nutrient starvation, and low oxygen tension, aiming to mimic facets of the intracellular lifestyle of provided initial clues to proteins relevant for intracellular survival and manipulation of the host cell. Further attempts were aimed at identifying the biological functions of the hypothetical proteins, which still make up a quarter of the gene products of , and at characterizing posttranslational modifications. Recent technological advances in MS have given rise to new methods such as selected reaction monitoring (SRM) and data-independent acquisition (DIA). These targeted, cutting-edge techniques combined with a public database of specific MS assays covering the entire proteome of allow the simple and reliable detection of any mycobacterial protein. Most recent studies attempt not only to identify but also to quantify absolute amounts of single proteins in the complex background of host cells without prior sample fractionation or enrichment. Finally, we will discuss the potential of proteomics to advance vaccinology, drug discovery, and biomarker identification to improve intervention and prevention measures for tuberculosis.

  • Citation: Gengenbacher M, Mouritsen J, Schubert O, Aebersold R, Kaufmann S. 2014. in the Proteomics Era. Microbiol Spectrum 2(2):MGM2-0020-2013. doi:10.1128/microbiolspec.MGM2-0020-2013.

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/content/journal/microbiolspec/10.1128/microbiolspec.MGM2-0020-2013
2014-03-21
2019-10-22

Abstract:

The emerging field of proteomics has contributed greatly to improving our understanding of the human pathogen over the last two decades. In this chapter we provide a comprehensive overview of mycobacterial proteome research and highlight key findings. First, studies employing a combination of two-dimensional gel electrophoresis and mass spectrometry (MS) provided insights into the proteomic composition, initially of the whole bacillus and subsequently of subfractions, such as the cell wall, cytosol, and secreted proteins. Comparison of results obtained under various culture conditions, i.e., acidic pH, nutrient starvation, and low oxygen tension, aiming to mimic facets of the intracellular lifestyle of provided initial clues to proteins relevant for intracellular survival and manipulation of the host cell. Further attempts were aimed at identifying the biological functions of the hypothetical proteins, which still make up a quarter of the gene products of , and at characterizing posttranslational modifications. Recent technological advances in MS have given rise to new methods such as selected reaction monitoring (SRM) and data-independent acquisition (DIA). These targeted, cutting-edge techniques combined with a public database of specific MS assays covering the entire proteome of allow the simple and reliable detection of any mycobacterial protein. Most recent studies attempt not only to identify but also to quantify absolute amounts of single proteins in the complex background of host cells without prior sample fractionation or enrichment. Finally, we will discuss the potential of proteomics to advance vaccinology, drug discovery, and biomarker identification to improve intervention and prevention measures for tuberculosis.

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in the Proteomics Era
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Citation: Gengenbacher M, Mouritsen J, Schubert O, Aebersold R, Kaufmann S. 2014. in the proteomics era. microbiolspec 2(2): doi:10.1128/microbiolspec.MGM2-0020-2013
/content/microbiolspec/10.1128/microbiolspec.MGM2-0020-2013.fig1
FIGURE 1
Functional annotation and abundance distribution of the proteome. Distribution of functional classes of the proteome as annotated in TubercuList v2.6 release 27 (March 2013), updated with functional annotation for many of the “conserved hypotheticals” and “unknowns” ( 4 ). Distribution of SRM-based absolute label-free abundance estimates for 2,195 proteins of H37Rv in a 1:1 mix of exponential and stationary cultures in rich medium ( 25 ). The concentration is given in femtomoles per microgram of extracted protein.
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FIGURE 1

Functional annotation and abundance distribution of the proteome. Distribution of functional classes of the proteome as annotated in TubercuList v2.6 release 27 (March 2013), updated with functional annotation for many of the “conserved hypotheticals” and “unknowns” ( 4 ). Distribution of SRM-based absolute label-free abundance estimates for 2,195 proteins of H37Rv in a 1:1 mix of exponential and stationary cultures in rich medium ( 25 ). The concentration is given in femtomoles per microgram of extracted protein.

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FIGURE 2
Technology-advanced proteomics. Early approaches identified only a small number of proteins. Combination of 2D-GE and untargeted shotgun mass spectrometry (MS), as well as extensive prefractionation of proteins or peptides, improved identification rates significantly. The latest targeted proteomics techniques, namely SRM, demonstrated identification of virtually all expressed proteins at given states in unfractionated cultures ( 25 ).
microbiolspec/2/2/MGM2-0020-2013-fig2_thmb.gif
microbiolspec/2/2/MGM2-0020-2013-fig2.gif
Image of FIGURE 2

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FIGURE 2

Technology-advanced proteomics. Early approaches identified only a small number of proteins. Combination of 2D-GE and untargeted shotgun mass spectrometry (MS), as well as extensive prefractionation of proteins or peptides, improved identification rates significantly. The latest targeted proteomics techniques, namely SRM, demonstrated identification of virtually all expressed proteins at given states in unfractionated cultures ( 25 ).

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FIGURE 3
Uncovering the proteome. Most of the current knowledge on proteomics has been generated by comparison of bacterial cultures, i.e., rich medium versus hypoxia or nutrient deprivation, conditions that might experience . Subfractions such as culture supernatant, cell wall debris, or the bacterial cytosol were separated by 2D-GE and subsequently analyzed by MS techniques. Study of the proteome during infection remained difficult: Due to the overwhelming protein abundance of the host as compared to the pathogen, enrichment for bacterial fractions was required prior to analysis by 2D-GE and shotgun MS. With the availability of the complete proteome libraries for ( 25 ) and the human host (U. Kusebauch et al., in preparation), SRM will allow simultaneous proteome analysis of the pathogen and the host in complex mixtures.
microbiolspec/2/2/MGM2-0020-2013-fig3_thmb.gif
microbiolspec/2/2/MGM2-0020-2013-fig3.gif
Image of FIGURE 3

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FIGURE 3

Uncovering the proteome. Most of the current knowledge on proteomics has been generated by comparison of bacterial cultures, i.e., rich medium versus hypoxia or nutrient deprivation, conditions that might experience . Subfractions such as culture supernatant, cell wall debris, or the bacterial cytosol were separated by 2D-GE and subsequently analyzed by MS techniques. Study of the proteome during infection remained difficult: Due to the overwhelming protein abundance of the host as compared to the pathogen, enrichment for bacterial fractions was required prior to analysis by 2D-GE and shotgun MS. With the availability of the complete proteome libraries for ( 25 ) and the human host (U. Kusebauch et al., in preparation), SRM will allow simultaneous proteome analysis of the pathogen and the host in complex mixtures.

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FIGURE 4
The Mtb Proteome Library. Proteome mapping: After harvesting bacterial cultures, proteins were extracted and digested with the proteolytic enzyme trypsin. The resulting peptides were fractionated using off-gel isoelectric focusing to reduce sample complexity, and each fraction was analyzed by shotgun MS. The peptide and protein identifications, as well as the corresponding spectra, can be browsed interactively in the PeptideAtlas database (http://www.PeptideAtlas.org). Proteome Library generation: From the collected data, the most MS-suited, unique peptides were selected for every annotated protein of . For proteins that had never been observed previously, representative peptides were predicted. The peptides were synthesized, pooled, and analyzed in SRM-triggered MS2 mode (SRM-MS2). From the resulting spectra the most intense fragment ions, as well as the chromatographic retention times, can be extracted. These MS coordinates, called SRM assays, constitute the synthetic Mtb Proteome Library and can be downloaded from the SRMAtlas database (http://www.SRMAtlas.org). Proteome Library validation: The SRM assays in the synthetic Mtb Proteome Library were validated for the detection of proteins in unfractionated mycobacterial lysates by SRM. The resulting quantitative SRM traces and statistical scores can be viewed in the PASSEL database (http://www.PeptideAtlas.org/passel). Reprinted from reference 25 with permission from Elsevier.
microbiolspec/2/2/MGM2-0020-2013-fig4_thmb.gif
microbiolspec/2/2/MGM2-0020-2013-fig4.gif
Image of FIGURE 4

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FIGURE 4

The Mtb Proteome Library. Proteome mapping: After harvesting bacterial cultures, proteins were extracted and digested with the proteolytic enzyme trypsin. The resulting peptides were fractionated using off-gel isoelectric focusing to reduce sample complexity, and each fraction was analyzed by shotgun MS. The peptide and protein identifications, as well as the corresponding spectra, can be browsed interactively in the PeptideAtlas database (http://www.PeptideAtlas.org). Proteome Library generation: From the collected data, the most MS-suited, unique peptides were selected for every annotated protein of . For proteins that had never been observed previously, representative peptides were predicted. The peptides were synthesized, pooled, and analyzed in SRM-triggered MS2 mode (SRM-MS2). From the resulting spectra the most intense fragment ions, as well as the chromatographic retention times, can be extracted. These MS coordinates, called SRM assays, constitute the synthetic Mtb Proteome Library and can be downloaded from the SRMAtlas database (http://www.SRMAtlas.org). Proteome Library validation: The SRM assays in the synthetic Mtb Proteome Library were validated for the detection of proteins in unfractionated mycobacterial lysates by SRM. The resulting quantitative SRM traces and statistical scores can be viewed in the PASSEL database (http://www.PeptideAtlas.org/passel). Reprinted from reference 25 with permission from Elsevier.

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FIGURE 5
SRM assay specificity in mycobacteria or host background. The theoretical specificity of SRM assays determined by the SRMCollider algorithm is shown as a cumulative plot of the number of peptides that can be uniquely identified with a given number of peptide-fragment ion pairs selected with decreasing intensity. Only background peptides with a chromatographic retention time close to that of the target peptide are considered as interfering background. The solid line indicates mycobacterial background. The dashed line indicates human background. Reprinted from reference 25 with permission from Elsevier.
microbiolspec/2/2/MGM2-0020-2013-fig5_thmb.gif
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FIGURE 5

SRM assay specificity in mycobacteria or host background. The theoretical specificity of SRM assays determined by the SRMCollider algorithm is shown as a cumulative plot of the number of peptides that can be uniquely identified with a given number of peptide-fragment ion pairs selected with decreasing intensity. Only background peptides with a chromatographic retention time close to that of the target peptide are considered as interfering background. The solid line indicates mycobacterial background. The dashed line indicates human background. Reprinted from reference 25 with permission from Elsevier.

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Tables

in the Proteomics Era
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Citation: Gengenbacher M, Mouritsen J, Schubert O, Aebersold R, Kaufmann S. 2014. in the proteomics era. microbiolspec 2(2): doi:10.1128/microbiolspec.MGM2-0020-2013
/content/microbiolspec/10.1128/microbiolspec.MGM2-0020-2013.T1
Table 1
Potential of proteomics for design of TB intervention measures
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Table 1

Potential of proteomics for design of TB intervention measures

Source: microbiolspec March 2014 vol. 2 no. 2 doi:10.1128/microbiolspec.MGM2-0020-2013

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