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Application of Two-Dimensional Difference Gel Electrophoresis in Identification of Factors Responsible for Virulence of Staphylococcus aureus

Part of the Methods in Molecular Biology book series (MIMB,volume 2069)

Abstract

Staphylococcus aureus is a dangerous opportunistic pathogen of humans and animals. Highly virulent and multi-antibiotic-resistant strains are of particular concern due to high invasiveness and limited array of useful treatment options. Proteomics allows identification and investigation of staphylococcal virulence factors to better understand and treat the related disease. Two-dimensional difference gel electrophoresis (2D DIGE) is a powerful method for identification of differences in staphylococcal proteomes, both intracellular and secretory. Not only the presence of particular proteins and their quantities may be determined, but also each modification changing the molecular mass and/or isoelectric point of a protein is trackable. Especially, 2D DIGE allows for detection of posttranslational modifications, including processing and degradation by proteases. For differential analysis, protein samples are labeled with spectrally distinguishable fluorescent dyes, mixed and separated according to their isoelectric point (first dimension), and then electrophoresed in the presence of sodium dodecyl sulfate according to their molecular mass (second dimension). Exceptional resolution of 2D DIGE allows to obtain focused and sharp protein spots, and identify a large number of differentiating proteins. Here we provide protocols for TRI Reagent-based preparation of high-quality samples for 2D DIGE, sample separation, and ways of handling differentiating protein spots which lead to samples ready for protein identification using MS.

Key words

  • 2D DIGE
  • Protein
  • Proteomics
  • Staphylococcus aureus
  • Virulence
  • Proteome
  • Electrophoresis

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Abbreviations

2D DIGE:

Two-dimensional difference gel electrophoresis

IEF:

Isoelectrofocusing

IPG:

Immobilized pH gradient

MS:

Mass spectrometry

PAGE:

Polyacrylamide gel electrophoresis

Rpm:

Revolutions per minute

RT:

Room temperature

SDS:

Sodium dodecyl sulfate

References

  1. Rasigade JP, Vandenesch F (2014) Staphylococcus aureus: a pathogen with still unresolved issues. Infect Genet Evol 21:510–514

    CAS  CrossRef  Google Scholar 

  2. Tong SYC, Davis JS, Eichenberger E, Holland TL, Fowler VG Jr (2015) Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev 28(3):603–661

    CAS  CrossRef  Google Scholar 

  3. Bonar EA, Bukowski M, Hydzik M, Jankowska U, Kedracka-Krok S, Groborz M et al (2018) Joint genomic and proteomic analysis identifies meta-trait characteristics of virulent and non-virulent Staphylococcus aureus strains. Front Cell Infect Microbiol 8:1–16. https://doi.org/10.3389/fcimb.2018.00313

    CrossRef  Google Scholar 

  4. Bonar E, Wojcik I, Jankowska U, Kedracka-Krok S, Bukowski M, Polakowska K et al (2016) Identification of secreted exoproteome fingerprints of highly-virulent and non-virulent Staphylococcus aureus strains. Front Cell Infect Microbiol 6:51. https://doi.org/10.3389/fcimb.2016.00051

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  5. Bonar E, Wójcik I, Wladyka B (2015) Proteomics in studies of Staphylococcus aureus virulence. Acta Biochim Pol 62(3):367–381

    CAS  CrossRef  Google Scholar 

  6. Hecker M, Mäder U, Völker U (2018) From the genome sequence via the proteome to cell physiology—pathoproteomics and pathophysiology of Staphylococcus aureus. Int J Med Microbiol 308(6):545–557

    CAS  CrossRef  Google Scholar 

  7. Arentz G, Weiland F, Oehler MK, Hoffmann P (2015) State of the art of 2D DIGE. Proteomics Clin Appl 9(3–4):277–288

    CAS  CrossRef  Google Scholar 

  8. Timms JF, Cramer R (2008) Difference gel electrophoresis. Proteomics 8(23–24):4886–4897

    CAS  CrossRef  Google Scholar 

  9. Pasquali M, Serchi T, Planchon S, Renaut J (2017) 2D-DIGE in proteomics. Methods Mol Biol 1654:245–254

    CAS  CrossRef  Google Scholar 

  10. Meleady P (2018) Two-dimensional gel electrophoresis and 2D-DIGE. Methods Mol Biol 1664:3–14

    CAS  CrossRef  Google Scholar 

  11. Koontz L (2014) TCA precipitation. Methods Enzymol 541:3–10

    CAS  CrossRef  Google Scholar 

  12. Wessel D, Flügge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138(1):141–143

    CAS  CrossRef  Google Scholar 

  13. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    CAS  CrossRef  Google Scholar 

  14. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    CAS  CrossRef  Google Scholar 

  15. Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68:850–858

    CAS  CrossRef  Google Scholar 

  16. Kang Y, Techanukul T, Mantalaris A, Mantalaris A, Nagy JM (2009) Comparison of three commercially available DIGE analysis software packages: minimal user intervention in gel-based proteomics. J Proteome Res 8(2):1077–1084. https://doi.org/10.1021/pr800588f

    CAS  CrossRef  PubMed  Google Scholar 

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Acknowledgments

Research on S. aureus using proteomics was supported by the National Science Centre (NCN, Poland) decision DEC-2014/13/B/NZ1/00043 (to BW), and by funds from IMPULS Program (to EB). The Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University is a partner of the Leading National Research Center (KNOW), which is supported by the Ministry of Science and Higher Education, Warsaw, Poland.

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Correspondence to Benedykt Wladyka .

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Bonar, E., Chlebicka, K., Dubin, G., Wladyka, B. (2020). Application of Two-Dimensional Difference Gel Electrophoresis in Identification of Factors Responsible for Virulence of Staphylococcus aureus. In: Ji, Y. (eds) Methicillin-Resistant Staphylococcus Aureus (MRSA) Protocols. Methods in Molecular Biology, vol 2069. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9849-4_11

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  • DOI: https://doi.org/10.1007/978-1-4939-9849-4_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9848-7

  • Online ISBN: 978-1-4939-9849-4

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