Abstract
This chapter describes a complete procedure for obtaining protein fingerprints of microorganisms using capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF). Staphylococcus aureus, a human pathogen responsible of frequent and resistant infections, is used as model microorganism to show the feasibility of this procedure. Bacteria are grown in different culture media or submitted to temperature or nitrosative stress conditions. After the growth of the bacteria, the protein extracts are obtained by cell lysis using sonication. The water-soluble fraction of these lysates is derivatized on-capillary with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde. The fluorescent products are analyzed by CE and detected by LIF. Practical advices for the interpretation of the electropherograms are given. To do so, the variations of the protein fingerprints of the bacteria with the culture conditions, such as growth medium, or the stressing conditions, such as heat shock or nitrosative stress, are used as example.
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References
Armstrong DW, Schulte G, Schneiderheinze JM et al (1999) Separating microbes in the manner of molecules. 1. Capillary Āelectrokinetic approaches. Anal Chem 71:5465ā5469
Petr J, Maier V (2012) Analysis of microorganisms by capillary electrophoresis. Trends Anal Chem 31:9ā22
Shen Y, Xiang F, Veenstra TD et al (1999) High-resolution capillary isoelectric focusing of complex protein mixtures from lysates of microorganisms. Anal Chem 71:5348ā5353
Hu S, Lee R, Zhang Z et al (2001) Protein analysis of an individual Caenorhabditis elegans single-cell embryo by capillary electrophoresis. J Chromatogr B 752:307ā310
Zhang Z, Krylov S, Arriaga EA et al (2000) One-dimensional protein analysis of an HT29 human colon adenocarcinoma cell. Anal Chem 72:318ā322
Hu S, Jiang J, Cook LM et al (2002) Capillary sodium dodecyl sulfate-DALT electrophoresis with laser-induced fluorescence detection for size-based analysis of proteins in human colon cancer cells. Electrophoresis 23:3136ā3142
Benito I, Marina ML, Saz JM et al (1999) Detection of bovine whey proteins by on-column derivatization capillary electrophoresis with laser-induced fluorescence monitoring. J Chromatogr A 841:105ā114
Csapo Z, Gerstner A, Sasvari-Szekely M et al (2000) Automated ultra-thin-layer SDS gel electrophoresis of proteins using non-covalent fluorescent labeling. Anal Chem 72:2519ā2525
Smith RD, Pasa-Tolic L, Lipton MS et al (2001) Rapid quantitative measurements of proteomes by Fourier transform ion cyclotron resonance mass spectrometry. Electrophoresis 22:1652ā1668
VanBolegen RA, Abshire KZ, Pertsemlidis A et al (1999) Gene-protein database of Escherichia coli K-12, edition 6. In: Neihardt FC, Curtiss R III, Ingraham JL et al (eds) Escherichia coli and Salmonella: cellular and molecular biology, 2nd edn. ASM Press, Washington, pp 2067ā2116
Ohta T, Honda K, Kuroda M et al (1993) Molecular characterization of the gene operon of heat-shock proteins hsp60 and hsp10 in methicillin-resistant Staphylococcus-aureus. BiocĀhem Biophys Res Commun 193:730ā737
Laport MS, de Castro ACD, Villardo A et al (2001) Expression of the major heat shock proteins DnaK and GroEL in Streptococcus pyogenes: a comparison to Enterococcus faecalis and Staphylococcus aureus. Curr Microbiol 42:264ā268
Katzif S, Danavall D, Bowers S et al (2003) The major cold shock gene, cspA, is involved in the susceptibility of Staphylococcus aureus to an antimicrobial peptide of human cathepsin G. Infect Immun 71:4304ā4312
Richardson AR, Dunman PM, Fang FC (2006) The nitrosative stress response of Staphylococcus aureus is required for resistance to innate immunity. Mol Microbiol 61:927ā939
Hecker M, Engelmann S, Cordwell SJ (2003) Proteomics of Staphylococcus aureusācurrent state and future challenges. J Chromatogr B 787:179ā195
Fuchs S, Pane-Farre J, Kohler C et al (2007) Anaerobic gene expression in Staphylococcus aureus. J Bacteriol 189:4275ā4289
Madigan MT, Martinko JM, Parker J (2003) Brock biology of microorganisms. Prentice Hall, Upper Saddle River, NJ
Zhang Z, Carpenter E, Puyan X et al (2001) Manipulation of protein fingerprints during on-column fluorescent labeling: protein fingerprinting of six Staphylococcus species by capillary electrophoresis. Electrophoresis 22:1127ā1132
Veledo MT, Pelaez-Lorenzo C, Gonzalez R et al (2010) Protein fingerprinting of Staphylococcus species by capillary electrophoresis with on-capillary derivatization and laser-induced fluorescence detection. Anal Chim Acta 658:81ā86
Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 72:248ā254
Veledo MT, de Frutos M, Diez-Masa JC (2005) Amino acids determination using capillary electrophoresis with on-capillary derivatization and laser-induced fluorescence detection. J Chromatogr A 1079:335ā343
Pelaez-Lorenzo C, Diez-Masa JC, Vasallo I et al (2009) A new sample preparation method compatible with capillary electrophoresis and laser-induced fluorescence for improving detection of low levels of Ī²-lactoglobulin in infant foods. Anal Chim Acta 649:202ā210
Veledo MT, de Frutos M, Diez-Masa JC (2005) Development of a method for quantitative analysis of the major whey proteins by capillary electrophoresis with on-capillary derivatization and laser-induced fluorescence detection. J Sep Sci 28:935ā940
DeGroote MA, Fang FC (1995) NO Inhibitions: antimicrobial properties of nitric oxide. Clin Infect Dis 21(Suppl 2):S162
Malawista SE, Montgomery RR, Van Blaricom G (1992) Evidence for reactive nitrogen intermediates in killing of staphylococci by human neutrophil cytoplasts. J Clin Invest 90:631ā636
Kaplan SS, Lancaster JR, Basford RE et al (1996) Effect of nitric oxide on staphylococcal killing and interactive effect with superoxide. Infect Immun 64:69ā76
Griess P (1879) Bemerkungen zu der Abhandlung der HH. Weselsky und Benedikt, āUber einige Azoverbindungenā. Ber Deutsch Chem Ges 12:426ā428
Davies KM, Wink DA, Saavedra JE et al (2001) Chemistry of the diazeniumdiolates. 2. Kinetics and mechanism of dissociation to nitric oxide in aqueous solution. J Am Chem Soc 123:5473ā5481
Acknowledgements
The Spanish Ministry of Science and Innovation (Project CTQ2009-09399), CSIC (Project 2010JP0013), and Domingo Martinez Foundation are acknowledged for financial support. Dr. A. Martinez (Department of Immunology, Hospital de la Princesa, Madrid, Spain) and Dr. A. Puerta (Institute of Organic Chemistry, CSIC, Spain) are acknowledged for helpful discussion, and Dr. J.M. Barcenilla (Institute of Food Science, Technology, and Nutrition, CSIC, Spain) for technical assistance.
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Pelaez-Lorenzo, C., Veledo, M.T., Gonzalez, R., de Frutos, M., Diez-Masa, J.C. (2013). Protein Fingerprinting of Staphylococcus aureus by Capillary Electrophoresis with On-Capillary Derivatization and Laser-Induced Fluorescence Detection. In: Volpi, N., Maccari, F. (eds) Capillary Electrophoresis of Biomolecules. Methods in Molecular Biology, vol 984. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-296-4_17
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DOI: https://doi.org/10.1007/978-1-62703-296-4_17
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