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Quantitative Metaproteomics: Functional Insights into Microbial Communities

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Environmental Microbiology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1096))

Abstract

Quantitative metaproteomics aims to accurately determine the relative abundances of thousands of proteins in a microbial community. This approach can be used to provide a comprehensive view of metabolic activities of organisms in microbial communities and uncover significant changes in protein expression between communities at different developmental stages, environment types or in response to different perturbations. Here, we describe three strategies for quantitative metaproteomics, including label-free, 15N metabolic labeling, and isobaric chemical labeling. The measurements are all based on a shotgun proteomics workflow involving proteolysis, two-dimensional liquid chromatogram-tandem mass spectrometry, and database searching against a metagenomic protein database. Quantitative metaproteomics was established and demonstrated using a model microbial community from acid mine drainage.

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References

  1. Tyson GW, Chapman J, Hugenholtz P, Allen EE, Ram RJ, Richardson PM, Solovyev VV, Rubin EM, Rokhsar DS, Banfield JF (2004) Community structure and metabolism through reconstruction of microbial genomes from the environment. Nature 428(6978): 37–43

    Article  CAS  PubMed  Google Scholar 

  2. Ram RJ, Verberkmoes NC, Thelen MP, Tyson GW, Baker BJ, Blake RC 2nd, Shah M, Hettich RL, Banfield JF (2005) Community proteomics of a natural microbial biofilm. Science 308(5730):1915–1920

    Article  CAS  PubMed  Google Scholar 

  3. Hettich RL, Pan C, Chourey K, Giannone RJ (2013) Metaproteomics: harnessing the power of high performance mass spectrometry to identify the suite of proteins that control metabolic activities in microbial communities. Anal Chem 85(9):4203–4214

    Article  CAS  PubMed  Google Scholar 

  4. Link AJ, Eng J, Schieltz DM, Carmack E, Mize GJ, Morris DR, Garvik BM, Yates JR 3rd (1999) Direct analysis of protein complexes using mass spectrometry. Nat Biotechnol 17(7):676–682

    Article  CAS  PubMed  Google Scholar 

  5. Eng JK, Mccormack AL, Yates JR (1994) An approach to correlate tandem mass-spectral data of peptides with amino-acid-sequences in a protein database. J Am Soc Mass Spectrom 5(11):976–989

    Article  CAS  PubMed  Google Scholar 

  6. Lo I, Denef VJ, Verberkmoes NC, Shah MB, Goltsman D, DiBartolo G, Tyson GW, Allen EE, Ram RJ, Detter JC, Richardson P, Thelen MP, Hettich RL, Banfield JF (2007) Strain-resolved community proteomics reveals recombining genomes of acidophilic bacteria. Nature 446(7135):537–541

    Article  CAS  PubMed  Google Scholar 

  7. Justice NB, Pan C, Mueller R, Spaulding SE, Shah V, Sun CL, Yelton AP, Miller CS, Thomas BC, Shah M, VerBerkmoes N, Hettich R, Banfield JF (2012) Heterotrophic archaea contribute to carbon cycling in low-pH, suboxic biofilm communities. Appl Environ Microbiol 78(23):8321–8330

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Liu H, Sadygov RG, Yates JR 3rd (2004) A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal Chem 76(14):4193–4201

    Article  CAS  PubMed  Google Scholar 

  9. VerBerkmoes NC, Denef VJ, Hettich RL, Banfield JF (2009) Systems biology: functional analysis of natural microbial consortia using community proteomics. Nat Rev Microbiol 7(3):196–205

    Article  CAS  PubMed  Google Scholar 

  10. Belnap CP, Pan C, Verberkmoes NC, Power ME, Samatova NF, Carver RL, Hettich RL, Banfield JF (2010) Cultivation and quantitative proteomic analyses of acidophilic microbial communities. ISME J 4:520–530

    Article  CAS  PubMed  Google Scholar 

  11. Belnap CP, Pan C, Denef VJ, Samatova NF, Hettich RL, Banfield JF (2011) Quantitative proteomic analyses of the response of acidophilic microbial communities to different pH conditions. ISME J 5(7):1152–1161

    Article  CAS  PubMed  Google Scholar 

  12. Mueller RS, Dill BD, Pan C, Belnap CP, Thomas BC, VerBerkmoes NC, Hettich RL, Banfield JF (2011) Proteome changes in the initial bacterial colonist during ecological succession in an acid mine drainage biofilm community. Environ Microbiol 13(8): 2279–2292

    Article  CAS  PubMed  Google Scholar 

  13. Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S, Purkayastha S, Juhasz P, Martin S, Bartlet-Jones M, He F, Jacobson A, Pappin DJ (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3(12):1154–1169

    Article  CAS  PubMed  Google Scholar 

  14. Thompson A, Schafer J, Kuhn K, Kienle S, Schwarz J, Schmidt G, Neumann T, Johnstone R, Mohammed AK, Hamon C (2003) Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem 75(8): 1895–1904

    Article  CAS  PubMed  Google Scholar 

  15. Wisniewski JR, Zougman A, Nagaraj N, Mann M (2009) Universal sample preparation method for proteome analysis. Nat Methods 6(5):359–362

    Article  CAS  PubMed  Google Scholar 

  16. Washburn MP, Wolters D, Yates JR 3rd (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 19(3):242–247

    Article  CAS  PubMed  Google Scholar 

  17. McDonald WH, Ohi R, Miyamoto DT, Mitchison TJ, Yates JR (2002) Comparison of three directly coupled HPLC MS/MS strategies for identification of proteins from complex mixtures: single-dimension LC-MS/MS, 2-phase MudPIT, and 3-phase MudPIT. Int J Mass Spectrom 219(1):245–251

    Article  CAS  Google Scholar 

  18. Pan C, Fischer CR, Hyatt D, Bowen BP, Hettich RL, Banfield JF (2011) Quantitative tracking of isotope flows in proteomes of microbial communities. Mol Cell Proteomics 10(4):M110.006049

    Article  PubMed  Google Scholar 

  19. Hyatt D, Pan C (2012) Exhaustive database searching for amino acid mutations in proteomes. Bioinformatics 28(14):1895–1901

    Article  CAS  PubMed  Google Scholar 

  20. Wang Y, Ahn TH, Li Z, Pan C (2013) Sipros/ProRata: a versatile informatics system for quantitative community proteomics. Bioinformatics 29(16):2064–2065

    Article  CAS  PubMed  Google Scholar 

  21. Pan C, Kora G, McDonald WH, Tabb DL, VerBerkmoes NC, Hurst GB, Pelletier DA, Samatova NF, Hettich RL (2006) ProRata: a quantitative proteomics program for accurate protein abundance ratio estimation with confidence interval evaluation. Anal Chem 78(20):7121–7131

    Article  CAS  PubMed  Google Scholar 

  22. Pan C, Kora G, Tabb DL, Pelletier DA, McDonald WH, Hurst GB, Hettich RL, Samatova NF (2006) Robust estimation of peptide abundance ratios and rigorous scoring of their variability and bias in quantitative shotgun proteomics. Anal Chem 78(20): 7110–7120

    Article  CAS  PubMed  Google Scholar 

  23. Li Z, Adams RM, Chourey K, Hurst GB, Hettich RL, Pan C (2012) Systematic comparison of label-free, metabolic labeling, and isobaric chemical labeling for quantitative proteomics on LTQ Orbitrap Velos. J Proteome Res 11(3):1582–1590

    Article  CAS  PubMed  Google Scholar 

  24. Peng J, Elias JE, Thoreen CC, Licklider LJ, Gygi SP (2003) Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. J Proteome Res 2(1):43–50

    Article  CAS  PubMed  Google Scholar 

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Acknowledgement

We like to thank Zhou Li, Nathan C. VerBerkmoes, Robert L. Hettich, Christopher P. Belnap, and Nagiza F. Samatova for their contributions in the development of the methodology described here. This work was funded by the US Department of Energy, Office of Biological and Environmental Research, and Office of Advanced Scientific Computing Research. Oak Ridge National Laboratory is managed by UT-Battelle LLC for the Department of Energy.

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Authors and Affiliations

  1. Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA

    Chongle Pan

  2. Department of Earth and Planetary Science, Policy, and Management, University of California, Berkeley, CA, USA

    Jillian F. Banfield

  3. Department of Environmental Science, Policy, and Management, University of California, Berkeley, CA, USA

    Jillian F. Banfield

Authors
  1. Chongle Pan
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  2. Jillian F. Banfield
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Editor information

Editors and Affiliations

  1. Dept. Chemistry & Biomolecular Sciences, Macquarie University, Sydney, New South Wales, Australia

    Ian T. Paulsen

  2. School of Molecular Bioscience, University of Sydney, Camperdown, New South Wales, Australia

    Andrew J. Holmes

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Pan, C., Banfield, J.F. (2014). Quantitative Metaproteomics: Functional Insights into Microbial Communities. In: Paulsen, I., Holmes, A. (eds) Environmental Microbiology. Methods in Molecular Biology, vol 1096. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-712-9_18

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  • DOI: https://doi.org/10.1007/978-1-62703-712-9_18

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-711-2

  • Online ISBN: 978-1-62703-712-9

  • eBook Packages: Springer Protocols

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