Abstract
The proteomic approaches have considerably evolved over the past two decades. This opened the doors for larger scale and deeper explorations of cellular physiology. Like for other living organisms, using the tools of proteomics has undoubtedly improved knowledge about the foodborne pathogen Listeria monocytogenes. Among the different technologies and approaches permanently evolving in the field of proteomics, the 2-DE is an analytical separation method of choice to resolve thousands of proteins simultaneously in a single gel, allowing their quantification, the study of their posttranslational modifications and the understanding of their biological function. In this, 2-DE remains a perfectly complementary technique to the new high-throughput techniques such as shotgun proteomics approaches. Moreover, in order to gain in analysis depth and improve knowledge about the target of action and the function of proteins in relation to their subcellular location, it is necessary to explore more specifically the different subcellular proteomes. Thus, the subproteomic analyses became essential and dramatically increased these last years, particularly on proteins secreted into the extracellular milieu, named exoproteome, or on cell envelope proteins (cell wall and membrane proteins) which are involved in the interactions with the surrounding environment. Here, the extraction and separation of L. monocytogenes subproteomes are described based on cell fractionation and 2-DE techniques. This chapter gives a workflow to obtain the exoproteome, the intracellular proteome, the cell wall, and membrane proteomes of the Gram-positive bacterium L. monocytogenes. The different steps of 2-DE technology, composed of a first dimension based on the separation of proteins according to their charge, an equilibration step, then a second dimension based on the separation of proteins according to their mass, and finally the staining of proteins in the gel are detailed. Emerging technologies to extract the exoproteome or the cell surface proteome after enzymatic shaving and to analyze them by shotgun method are also discussed briefly.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Cordwell SJ (2006) Technologies for bacterial surface proteomics. Curr Opin Microbiol 9:320–329
Santoni V, Molloy M, Rabilloud T (2000) Membrane proteins and proteomics: un amour impossible? Electrophoresis 21:1054–1070
Santoni V, Kiefer S, Desclaux D, Masson F, Rabilloud T (2000) Membrane proteomics: use of additive main effects with multiplicative interaction model to classify plasma membrane proteins according to their solubility and electrophoretic properties. Electrophoresis 21:3329–3344
Mastro R, Hall M (1999) Protein delipidation and precipitation by tri-n-butylphosphate, acetone, and methanol treatment for isoelectric focusing and two-dimensional gel electrophoresis. Anal Biochem 273:313–315
Deshusses JMP, Burgess JA, Scherl A, Wenger Y, Walter N, Converset V, Paesano S, Corthals GL, Hochstrasser DF, Sanchez J-C (2003) Exploitation of specific properties of trifluoroethanol for extraction and separation of membrane proteins. Proteomics 3:1418–1424
Schaumburg J, Diekmann O, Hagendorff P, Bergmann S, Rohde M, Hammerschmidt S, Jansch L, Wehland J, Karst U (2004) The cell wall subproteome of Listeria monocytogenes. Proteomics 4:2991–3006
Pucciarelli MG, Calvo E, Sabet C, Bierne H, Cossart P, Garcia del Portillo F (2005) Identification of substrates of the Listeria monocytogenes sortases A and B by a non-gel proteomic analysis. Proteomics 5:4808–4817
Calvo E, Pucciarelli MG, Bierne H, Cossart P, Albar JP, Garcia del Portillo F (2005) Analysis of the Listeria cell wall proteome by two-dimensional nanoliquid chromatography coupled to mass spectrometry. Proteomics 5:433–443
Folio P, Chavant P, Chafsey I, Belkorchia A, Chambon C, Hebraud M (2004) Two-dimensional electrophoresis database of Listeria monocytogenes EGDe proteome and proteomic analysis of mid-log and stationary growth phase cells. Proteomics 4:3187–3201
Dumas E, Meunier B, Berdague J-L, Chambon C, Desvaux M, Hebraud M (2008) Comparative analysis of extracellular and intracellular proteomes of twelve Listeria monocytogenes strains: differential protein expression is correlated to serovar. Appl Environ Microbiol 74:7399–7409
Dumas E, Desvaux M, Chambon C, Hebraud M (2009) Insight into the core and variant exoproteomes of Listeria monocytogenes species by comparative subproteomic analysis. Proteomics 9:3136–3155
Renier S, Chambon C, Viala D, Chagnot C, Hebraud M, Desvaux M (2013) Exoproteomic analysis of the SecA2-dependent secretion in Listeria monocytogenes EGDe. J Proteomics 80:183–195
Olaya-Abril A, Jiménez-Munguía I, Gómez-Gascón L, Rodríguez-Ortega MJ (2013) Surfomics: shaving live organisms for a fast proteomic identification of surface proteins. J Proteomics 97:164–176
Tjalsma H, Lambooy L, Hermans PW, Swinkels KW (2008) Shedding & shaving: disclosure of proteomic expressions on a bacterial face. Proteomics 8:1416–1428
Solis N, Larsen MR, Cordwell SJ (2010) Improved accuracy of cell surface shaving proteomics in Staphylococcus aureus using a false-positive control. Proteomics 10:2037–2049
Dreisbach A, Hempel K, Buist G, Hecker M, Becher D, van Dijl JM (2010) Profiling the surfacome of Staphylococcus aureus. Proteomics 10:3082–3096
Bohle LA, Riaz T, Egge-Jacobsen W, Skaugen M, Busk OL, Eijsink VGH, Mathiesen G (2011) Identification of surface proteins in Enterococcus faecalis V583. BMC Genomics 12:135
Olaya-Abril A, Gómez-Gascón L, Jiménez-Munguía I, Obando I, Rodríguez-Ortega MJ (2012) Another turn of the screw in shaving Gram-positive bacteria: optimization of proteomics surface protein identification in Streptococcus pneumoniae. J Proteomics 75:3733–3746
Wei Z, Fu Q, Liu X, Xiao P, Lu Z, Chen Y (2013) Identification of Streptococcus equi ssp. zooepidemicus surface associated proteins by enzymatic shaving. Vet Microbiol 159:519–525
Neuhoff V, Stamm R, Eibl H (1985) Clear background and highly sensitive protein staining with coomassie blue dyes in polyacrylamide gels: a systematic analysis. Electrophoresis 6:427–448
Neuhoff V, Arold N, Taube D, Ehrhardt W (1988) Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9:255–262
Chevallet M, Santoni V, Poinas A, Rouquié D, Fuchs A, Kieffer S, Rossignol M, Lunardi J, Garin J, Rabilloud T (1998) New zwitterionic detergents improve the analysis of membrane proteins by two-dimensional electrophoresis. Electrophoresis 19:1901–1909
Tastet C, Charmont S, Chevallet M, Luche S, Rabilloud T (2003) Structure-efficiency relationships of zwitterionic detergents as protein solubilizers in two-dimensional electrophoresis. Proteomics 3:111–121
Planchon S, Chambon C, Desvaux M, Chafsey I, Leroy S, Talon R, Hebraud M (2007) Proteomic analysis of cell envelope from Staphylococcus xylosus C2a, a coagulase-negative Staphylococcus. J Res Proteome 6:3566–3580
Planchon S, Desvaux M, Chafsey I, Chambon C, Leroy S, Hebraud M, Talon R (2009) Comparative subproteome analyses of plaktonic and sessile Staphylococcus xylosus C2a: new insight in cell physiology of a coagulase-negative Staphylococcus in biofilm. J Res Proteome 8:1797–1809
Rabilloud T, Lelong C (2011) Two-dimensional gel electrophoresis in proteomics: a tutorial. J Proteomics 74:1829–1841
Acknowledgment
The author thanks I. Chafsey for her valuable technical expertise in proteomics approaches and its constantly renewed investment in the training of students to the different proteomics methodologies. The author also thanks C. Chambon, engineer on the proteomics component of the Metabolism Exploration Platform (PFEMcp) from INRA Clermont-Ferrand, who brings his skills to perform all our analysis by mass spectrometry with a remarkable efficiency and dedication and a constant concern to improve the methods and to optimize the analytical performances. Finally, I thank all my students who have contributed to the development and improvement of these techniques in the lab.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Hébraud, M. (2014). Analysis of Listeria monocytogenes Subproteomes. In: Jordan, K., Fox, E., Wagner, M. (eds) Listeria monocytogenes. Methods in Molecular Biology, vol 1157. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0703-8_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-0703-8_10
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0702-1
Online ISBN: 978-1-4939-0703-8
eBook Packages: Springer Protocols