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.
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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
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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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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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