Abstract
The mammalian proteomes are inherently complex, and therefore a global analysis of these proteomes presents a great technical challenge. It is recognized that there is no single method today that is able to probe an entire human proteome, and that a combination of several methodological approaches provides the most flexible strategy (1). To date, the most widely used methodology for proteome analysis involves a combination of two-dimensional gel electrophoresis (2-DE), mass spectrometry, and bioinformatics tools. Although this “classical” approach has been successfully applied in many studies, it suffers from a number of limitations, such as incomplete proteome coverage, low throughput, and so on (2). In order to overcome these limitations, a number of modifications of the 2-DE-based approach have been introduced, such as new strategies for sample pre-fractionation, introduction of medium- and narrow-range immobilized pH gradient (IPG) strips, development of fluorescent protein stains and differential gel electrophoresis, and design of new robotics systems for protein processing. In addition to modifications in the 2-DE-based proteomics, new methodologies were developed that utilize only one of the dimensions of 2-DE, i.e., isoelectric focusing (IEF) or sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). For example, several strategies that combine in-gel IEF with mass spectrometry (MS) were described, such as direct mass spectrometric characterization of intact proteins in IEF gels (3,4), or identification of proteins in selected regions of IEF gels via proteolytic digestion and mass spectrometry (5–7). Furthermore, in-solution IEF has been also been used for protein fractionation, and new devices for liquid IEF have been designed (8–12).
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Hancock, W. S., Wu, S. L., and Shieh, P. (2002) The challenges of developing a sound proteomics strategy. Proteomics 2, 352–359.
Beranova-Giorgianni, S. (2003) Proteome analysis by two-dimensional gel electrophoresis and mass spectrometry: strengths and limitations. Trends Anal. Chem. 22, 273–281.
Ogorzalek Loo, R. R., Stevenson, T. I., Mitchell, C., Loo, J. A., and Andrews, P. C. (1996) Mass spectrometry of proteins directly from polyacrylamide gels. Anal. Chem. 68, 1910–1917.
Loo, J. A., Brown, J., Critchley, G., Mitchell, C., Andrews, P. C., and Ogorzalek Loo, R. R. (1999) High sensitivity mass spectrometric methods for obtaining intact molecular weights from gel-separated proteins. Electrophoresis 20, 743–748.
Steinberg, T. H., Chernokalskaya, E., Berggren, K., et al. (2000) Ultrasensitive fluorescence protein detection in isoelectric focusing gels using a ruthenium metal chelate stain. Electrophoresis 21, 486–496.
Castellanos-Serra, L., Vallin, A., Proenza, W., Le Caer, J. P., and Rossier, J. (2001) An optimized procedure for detection of proteins on carrier ampholyte isoelectric focusing and immobilized pH gradient gels with imidazole and zinc salts: its application to the identification of isoelectric focusing separated isoforms by in-gel proteolysis and mass spectrometry analysis. Electrophoresis 22, 1677–1685.
Le Bihan, T., Pinto, D., and Figeys, D. (2001) Nanoflow gradient generator coupled with gm-LC-ESI-MS/MS for protein identification. Anal. Chem. 73, 1307–1315.
Zuo, X., and Speicher, D. W. (2000) A method for global analysis of complex proteomes using sample prefractionation by solution isoelectrofocusing prior to two-dimensional electrophoresis. Anal. Biochem. 284, 266–278.
Herbert, B. and Righetti, P. G. (2000) A turning point in proteome analysis: sample prefractionation via multicompartment electrolyzers with isoelectric membranes. Electrophoresis 21, 3639–3648.
Hoffman, P., Ji, H., Moritz, R. L., et al. (2001) Continuous free-flow electrophoresis separation of cytosolic proteins from the human colon carcinoma cell line LIM 1215: a non two-dimensional gel electrophoresis-based proteome analysis strategy. Proteomics 1, 807–818.
Ros, A., Faupel, M., Mees, H., et al. (2002) Protein purification by off-gel electrophoresis. Proteomics 2, 151–156.
Righetti, P. G., Castagna, A., Herbert, B., Reymond, F., and Rossier, J. S. (2003) Prefractionation techniques in proteome analysis. Proteomics 3, 1397–1407.
Giorgianni, F., Desiderio, D. M., and Beranova-Giorgianni, S. (2003) Proteome analysis using isoelectric focusing in immobilized pH gradient gels followed by mass spectrometry. Electrophoresis 24, 253–259.
Link, A. J., Eng, J., Schieltz, D. M., et al. (1999) Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol. 17, 676–682.
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© 2005 Humana Press Inc., Totowa, NJ
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Giorgianni, F., Beranova-Giorgianni, S. (2005). Analysis of the Proteomes in Human Tissues by In-Gel Isoelectric Focusing and Mass Spectrometry. In: Walker, J.M. (eds) The Proteomics Protocols Handbook. Springer Protocols Handbooks. Humana Press. https://doi.org/10.1385/1-59259-890-0:367
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DOI: https://doi.org/10.1385/1-59259-890-0:367
Publisher Name: Humana Press
Print ISBN: 978-1-58829-343-5
Online ISBN: 978-1-59259-890-8
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