Isolation and identification of Enterococcus faecalis membrane proteins using membrane shaving, 1D SDS/PAGE, and mass
Peter Cathro1, Peter McCarthy2, Peter Hoffmann3
1Oral Microbiology Laboratory School of Dentistry The University of Adelaide South Australia Australia.
Abstract:
Enterococcus faecalis is a significant nosocomial pathogen, which is able to survive in diverse environments and resist killing with antimicrobial therapies. The expression of cell membrane proteins play an important role in how bacteria respond to environmental stress. As such, the capacity to identify and study membrane protein expression is critical to our understanding of how specific proteins influence bacterial survival. Here, we describe a combined approach to identify membrane proteins of E. faecalis ATCC V583 using membranes fractionated by either 1D SDS/PAGE or membrane shaving, coupled with LC-ESI mass spectrometry. We identified 222 membrane-associated proteins, which represent approximately 24% of the predicted membrane-associated proteome: 170 were isolated using 1D SDS/PAGE and 68 with membrane shaving, with 36 proteins being common to both the techniques. Of the proteins identified by membrane shaving, 97% were membrane-associated with the majority being integral membrane proteins (89%). Most of the proteins identified with known physiology are involved with transportation across the membrane. The combined 1D SDS/PAGE and membrane shaving approach has produced the greatest number of membrane proteins identified from E. faecalis to date. These protocols will aid future researchers investigating changes in the membrane proteome of E. faecalis by improving our understanding of how E. faecalis adapts and responds to its environment.
Insights
This study identifies 222 membrane proteins in Enterococcus faecalis, a common hospital pathogen. The novel combined method enhances understanding of bacterial adaptation and antimicrobial resistance mechanisms.
Area of Science:
- Microbiology
- Proteomics
- Bacterial Pathogenesis
Background:
- Enterococcus faecalis is a significant nosocomial pathogen known for environmental adaptability and antimicrobial resistance.
- Cell membrane proteins are crucial for bacterial stress response and survival.
- Understanding E. faecalis membrane proteome is key to deciphering its survival strategies.
Purpose of the Study:
- To develop and apply a combined proteomic approach for identifying membrane proteins in E. faecalis.
- To characterize the identified membrane proteins and their functions.
- To establish protocols for future studies on E. faecalis membrane proteome dynamics.
Main Methods:
- Fractionation of E. faecalis ATCC V583 membranes using 1D SDS/PAGE and membrane shaving.
- Analysis of fractionated samples using Liquid Chromatography-Electrospray Ionization mass spectrometry (LC-ESI-MS).
- Identification and quantification of membrane-associated proteins.
Main Results:
- Identification of 222 membrane-associated proteins, representing approximately 24% of the predicted proteome.
- 170 proteins identified via 1D SDS/PAGE, 68 via membrane shaving, with 36 common to both.
- Membrane shaving yielded high-purity membrane proteins (97%), predominantly integral membrane proteins (89%).
- Identified proteins are largely involved in transmembrane transport.
Conclusions:
- The combined 1D SDS/PAGE and membrane shaving approach is highly effective for comprehensive membrane proteome analysis in E. faecalis.
- This study provides the most extensive list of identified membrane proteins in E. faecalis to date.
- The developed protocols will facilitate future research into E. faecalis adaptation and response to environmental challenges and antimicrobial therapies.
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