Skip to main content
SpringerLink
Log in

Fractionation and Analysis of Mycobacterial Proteins

  • Protocol
  • First Online:
Mycobacteria Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1285))

Abstract

The extraction and isolation of native bacterial proteins continue to be valuable technical pursuits in order to understand bacterial physiology, screen for virulence determinants, and describe antigens. In this chapter, methods for the manipulation of whole mycobacterial cells are described in detail. Specifically, the concentration of spent culture filtrate media is described in order to permit separation of soluble, secreted proteins; several discrete separation techniques, including precipitation of protein mixtures with ammonium sulfate and separation of proteins by hydrophobic chromatography are also provided. Similarly, the generation of whole cell lysate and facile separation of lysate into subcellular fractions to afford cell wall, cell membrane, and cytosol enriched proteins is described. Due to the hydrophobic nature of cell wall and cell membrane proteins, several extraction protocols to resolve protein subsets (such as extraction with urea and SDS) are also provided, as well as a separation technique (isoelectric focusing) that can be applied to separate hydrophobic proteins. Lastly, two commonly used analytical techniques, in-gel digestion of proteins for LC-MS and analysis of intact proteins by MALDI-ToF MS, are provided for rapid analysis of discrete proteins within subcellular or chromatographic fractions. While these methods were optimized for the manipulation of Mycobacterium tuberculosis cells, they have been successfully applied to extract and isolate Mycobacterium leprae, Mycobacterium ulcerans, and Mycobacterium avium proteins. In addition, a number of these methods may be applied to extract and analyze mycobacterial proteins from cell lines and host derived samples.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Delogu G, Manganelli R, Brennan MJ (2014) Critical research concepts in tuberculosis vaccine development. Clin Microbiol Infect 20(Suppl 5):59–65. doi:10.1111/1469-0691.12460

    Article  PubMed  Google Scholar 

  2. Liu L, Zhang WJ, Zheng J, Fu H, Chen Q, Zhang Z, Chen X, Zhou B, Feng L, Liu H, Jin Q (2014) Exploration of novel cellular and serological antigen biomarkers in the ORFeome of Mycobacterium tuberculosis. Mol Cell Proteomics 13(3):897–906. doi:10.1074/mcp.M113.032623

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Shekhawat SD, Jain RK, Gaherwar HM, Purohit HJ, Taori GM, Daginawala HF, Kashyap RS (2014) Heat shock proteins: possible biomarkers in pulmonary and extrapulmonary tuberculosis. Hum Immunol 75(2):151–158. doi:10.1016/j.humimm.2013.11.007

    Article  CAS  PubMed  Google Scholar 

  4. Wallis RS, Kim P, Cole S, Hanna D, Andrade BB, Maeurer M, Schito M, Zumla A (2013) Tuberculosis biomarkers discovery: developments, needs, and challenges. Lancet Infect Dis 13(4):362–372. doi:10.1016/S1473-3099(13)70034-3

    Article  PubMed  Google Scholar 

  5. Purohit MR, Mustafa T, Wiker HG, Sviland L (2012) Rapid diagnosis of tuberculosis in aspirate, effusions, and cerebrospinal fluid by immunocytochemical detection of Mycobacterium tuberculosis complex specific antigen MPT64. Diagn Cytopathol 40(9):782–791. doi:10.1002/dc.21637

    Article  PubMed  Google Scholar 

  6. Covert BA, Spencer JS, Orme IM, Belisle JT (2001) The application of proteomics in defining the T cell antigens of Mycobacterium tuberculosis. Proteomics 1(4):574–586

    Article  CAS  PubMed  Google Scholar 

  7. Wolfe LM, Mahaffey SB, Kruh NA, Dobos KM (2010) Proteomic definition of the cell wall of Mycobacterium tuberculosis. J Proteome Res 9(11):5816–5826. doi:10.1021/pr1005873

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. de Souza GA, Wiker HG (2011) A proteomic view of mycobacteria. Proteomics 11(15):3118–3127. doi:10.1002/pmic.201100043

    Article  PubMed  Google Scholar 

  9. Deenadayalan A, Heaslip D, Rajendiran AA, Velayudham BV, Frederick S, Yang H-L, Dobos K, Belisle JT, Raja A (2010) Immunoproteomic identification of human T cell antigens of Mycobacterium tuberculosis that differentiate healthy contacts from tuberculosis patients. Mol Cell Proteomics 9(3):538–549. doi:10.1074/mcp.M900299-MCP200

    Article  CAS  PubMed  Google Scholar 

  10. Lange S, Rosenkrands I, Stein R, Andersen P, Kaufmann SH, Jungblut PR (2014) Analysis of protein species differentiation among mycobacterial low-Mr-secreted proteins by narrow pH range Immobiline gel 2-DE-MALDI-MS. J Proteomics 97:235–244. doi:10.1016/j.jprot.2013.06.036

    Article  CAS  PubMed  Google Scholar 

  11. Sartain MJ, Slayden RA, Singh KK, Laal S, Belisle JT (2006) Disease state differentiation and identification of tuberculosis biomarkers via native antigen array profiling. Mol Cell Proteomics 5(11):2102–2113. doi:10.1074/mcp.M600089-MCP200

    Article  CAS  PubMed  Google Scholar 

  12. Kerns PW, Ackhart DF, Basaraba RJ, Leid JG, Shirtliff ME (2014) Mycobacterium tuberculosis pellicles express unique proteins recognized by the host humoral response. Pathog Dis 70(3):347–358. doi:10.1111/2049-632X.12142

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Giri PK, Kruh NA, Dobos KM, Schorey JS (2010) Proteomic analysis identifies highly antigenic proteins in exosomes from M. tuberculosis-infected and culture filtrate protein-treated macrophages. Proteomics 10(17):3190–3202. doi:10.1002/pmic.200900840

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Mattow J, Siejak F, Hagens K, Becher D, Albrecht D, Krah A, Schmidt F, Jungblut PR, Kaufmann SH, Schaible UE (2006) Proteins unique to intraphagosomally grown Mycobacterium tuberculosis. Proteomics 6(8):2485–2494. doi:10.1002/pmic.200500547

    Article  CAS  PubMed  Google Scholar 

  15. Lamont EA, Janagama HK, Ribeiro-Lima J, Vulchanova L, Seth M, Yang M, Kurmi K, Waters WR, Thacker T, Sreevatsan S (2014) Circulating Mycobacterium bovis peptides and host response proteins as biomarkers for unambiguous detection of subclinical infection. J Clin Microbiol 52(2):536–543. doi:10.1128/JCM.02433-13

    Article  PubMed  PubMed Central  Google Scholar 

  16. Kashino SS, Pollock N, Napolitano DR, Rodrigues V Jr, Campos-Neto A (2008) Identification and characterization of Mycobacterium tuberculosis antigens in urine of patients with active pulmonary tuberculosis: an innovative and alternative approach of antigen discovery of useful microbial molecules. Clin Exp Immunol 153(1):56–62. doi:10.1111/j.1365-2249.2008.03672.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Cheng Y, Schorey JS (2013) Exosomes carrying mycobacterial antigens can protect mice against Mycobacterium tuberculosis infection. Eur J Immunol 43(12):3279–3290. doi:10.1002/eji.201343727

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Ramachandra L, Qu Y, Wang Y, Lewis CJ, Cobb BA, Takatsu K, Boom WH, Dubyak GR, Harding CV (2010) Mycobacterium tuberculosis synergizes with ATP to induce release of microvesicles and exosomes containing major histocompatibility complex class II molecules capable of antigen presentation. Infect Immun 78(12):5116–5125. doi:10.1128/IAI.01089-09

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Prevots DR, Shaw PA, Strickland D, Jackson LA, Raebel MA, Blosky MA, Montes de Oca R, Shea YR, Seitz AE, Holland SM, Olivier KN (2010) Nontuberculous mycobacterial lung disease prevalence at four integrated health care delivery systems. Am J Respir Crit Care Med 182(7):970–976. doi:10.1164/rccm.201002-0310OC

    Article  PubMed  PubMed Central  Google Scholar 

  20. Wentworth AB, Drage LA, Wengenack NL, Wilson JW, Lohse CM (2013) Increased incidence of cutaneous nontuberculous mycobacterial infection, 1980 to 2009: a population-based study. Mayo Clin Proc 88(1):38–45. doi:10.1016/j.mayocp.2012.06.029

    Article  PubMed  Google Scholar 

  21. Dobos KM, Swiderek K, Khoo KH, Brennan PJ, Belisle JT (1995) Evidence for glycosylation sites on the 45-kilodalton glycoprotein of Mycobacterium tuberculosis. Infect Immun 63(8):2846–2853

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Mawuenyega KG, Forst CV, Dobos KM, Belisle JT, Chen J, Bradbury EM, Bradbury AR, Chen X (2005) Mycobacterium tuberculosis functional network analysis by global subcellular protein profiling. Mol Biol Cell 16(1):396–404. doi:10.1091/mbc.E04-04-0329

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Prados-Rosales R, Weinrick BC, Pique DG, Jacobs WR Jr, Casadevall A, Rodriguez GM (2014) Role for Mycobacterium tuberculosis membrane vesicles in iron acquisition. J Bacteriol 196(6):1250–1256. doi:10.1128/JB.01090-13, JB.01090-13 [pii]

    Article  PubMed  PubMed Central  Google Scholar 

  24. Hirschfield GR, McNeil M, Brennan PJ (1990) Peptidoglycan-associated polypeptides of Mycobacterium tuberculosis. J Bacteriol 172(2):1005–1013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Konigsberg WH, Henderson L (1983) Removal of sodium dodecyl sulfate from proteins by ion-pair extraction. Methods Enzymol 91:254–259

    Article  CAS  PubMed  Google Scholar 

  26. Radolf JD, Chamberlain NR, Clausell A, Norgard MV (1988) Identification and localization of integral membrane proteins of virulent Treponema pallidum subsp. pallidum by phase partitioning with the nonionic detergent triton X-114. Infect Immun 56(2):490–498

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Belisle JT, Vissa VD, Sievert T, Takayama K, Brennan PJ, Besra GS (1997) Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis. Science 276(5317):1420–1422

    Article  CAS  PubMed  Google Scholar 

  28. Hellman U, Wernstedt C, Gonez J, Heldin CH (1995) Improvement of an “In-Gel” digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Anal Biochem 224(1):451–455, S0003-2697(85)71070-6 [pii]

    Article  CAS  PubMed  Google Scholar 

  29. Rosenfeld J, Capdevielle J, Guillemot JC, Ferrara P (1992) In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal Biochem 203(1):173–179, 0003-2697(92)90061-B [pii]

    Article  CAS  PubMed  Google Scholar 

  30. Taylor JL, Wieczorek A, Keyser AR, Grover A, Flinkstrom R, Karls RK, Bielefeldt-Ohmann H, Dobos KM, Izzo AA (2012) HspX-mediated protection against tuberculosis depends on its chaperoning of a mycobacterial molecule. Immunol Cell Biol 90(10):945–954. doi:10.1038/icb.2012.34

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Wieczorek AE, Troudt JL, Knabenbauer P, Taylor J, Pavlicek RL, Karls R, Hess A, Davidson RM, Strong M, Bielefeldt-Ohmann H, Izzo AA, Dobos KM (2014) HspX vaccination and role in virulence in the guinea pig model of tuberculosis. Pathog Dis. doi:10.1111/2049-1632X.12147

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by NIH-NIAID Contracts HHSN266200400091c and ATCC contract 2010-0516-0005 (a subcontract of NIH-NIAID Contract HHSN272201000027c). The original work is credited to NIH-NIAID Contract AI-75320.

Author information

Authors and Affiliations

  1. Mycobacteria Research Laboratories, Department of Microbiology, Immunology, and Pathology, Colorado State University, 1682 Campus Delivery, Fort Collins, CO, 80523, USA

    Megan C. Lucas, Rachel M. Hazenfield, Nicole A. Kruh-Garcia & Karen M. Dobos

  2. Proteomics and Metabolomics Facility, Colorado State University, Fort Collins, CO, USA

    Lisa M. Wolfe

  3. Department of Clinical Sciences, Colorado State University, Fort Collins, CO, USA

    Jade Kurihara

  4. Mycobacteria Research Laboratories, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO, USA

    John Belisle

Authors
  1. Megan C. Lucas
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lisa M. Wolfe
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rachel M. Hazenfield
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jade Kurihara
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Nicole A. Kruh-Garcia
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. John Belisle
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Karen M. Dobos
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Karen M. Dobos .

Editor information

Editors and Affiliations

  1. Infectious Disease Research Inst (IDRI), Seattle, Washington, USA

    Tanya Parish

  2. Infectious Disease Research Inst (IDRI), Seattle, Washington, USA

    David M. Roberts

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Lucas, M.C. et al. (2015). Fractionation and Analysis of Mycobacterial Proteins. In: Parish, T., Roberts, D. (eds) Mycobacteria Protocols. Methods in Molecular Biology, vol 1285. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2450-9_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2450-9_4

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2449-3

  • Online ISBN: 978-1-4939-2450-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation