Location: Warmwater Aquaculture Research UnitTitle: Improved Solubilization of Surface Proteins from Listeria monocytogenes for Two-dimensional Gel Electrophoresis) Author
Submitted to: Electrophoresis
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/5/2007
Publication Date: 10/26/2007
Citation: Mujihid, S., Pechan, T., Wang, C. 2007. Improved Solubilization of Surface Proteins from Listeria monocytogenes for Two-dimensional Gel Electrophoresis. Electrophoresis. 28:3998-4007. Interpretive Summary: Two-dimensional gel electrophoresis (2-DE) is an extremely useful technique for protein analysis. However, solubilization of bacterial surface proteins, a step necessary for effective 2-DE, is particularly problematic for Gram-positive bacteria such as the foodborne pathogen L m. We developed a technique that allowed solubilization of L mon by treating with mutanolysin in combination with amidosulfobetaine-14 (ASB-14),urea, and thiourea. This treatment allowed successful solubilxation and 2-DE of L mon surface proteins and revealed 29 characterized proteins, 17 of which had not been previously reported. This technique will be useful for more efficient 2-DE of L. monocytogenes and other Gram-positive bacteria surface proteins which will improve our and lead to a better understanding and control of these bacteria.
Technical Abstract: Solubilization of bacterial surface (cell wall and membrane-associated) proteins for 2-DE is challenging, particularly in the case of Gram-positive bacteria. This is primarily due to strong protein association with the cell wall peptidoglycan and protein hydrophobicity. We solubilized surface proteins for 2-DE from the Gram-positive pathogen Listeria monocytogenes using mutanolysin, which digests cell wall peptidoglycan, and one of three different mixtures of zwitterionic detergent and chaotropes: (i) CHAPS/urea, (ii) amidosulfobetaine-14 (ASB-14)/urea/thiourea (iii) N-decyl-N,N'-dimethyl-3-ammonio-1-propanesulfonate/urea/thiourea. Cell lysis with mutanolysin followed by solubilization with ASB-14/urea/thiourea gave the highest overall protein yield with the best 2-DE resolution. Protein spot identification by MALDI-TOF/TOF-MS analysis revealed 29 characterized surface proteins of L. monocytogenes, 17 of which have not previously been reported on the surface proteome map. This is the first report describing the successful solubilization and 2-DE of L. monocytogenes proteins bound to the cell surface via an LPXTG motif or by a hydrophobic tail. The increase in surface proteome coverage obtained by mutanolysin and ASB-14/urea/thiourea solubilization suggests the utility of this method for future analytical and comparative studies of surface proteins from Listeria, and possibly other Gram-positive bacteria, using 2-DE proteomic analysis. An updated 2-DE reference map of L. monocytogenes surface proteins is presented.