在蛋白质组学研究策略的能力和局限的电泳分离.doc
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1、在蛋白质组学研究策略的能力和局限的电泳分离Power and limitations of electrophoretic separations in proteomics strategiesThierry. Rabilloud 1,2, Ali R.Vaezzadeh 3 , Noelle Potier 4, Ccile Lelong1,5, Emmanuelle Leize-Wagner 4, Mireille Chevallet 1,21: CEA, IRTSV, LBBSI, 38054 GRENOBLE, France.2: CNRS, UMR 5092, Biochimie e
2、t Biophysique des Systmes Intgrs, Grenoble France3: Biomedical Proteomics Research Group, Central Clinical Chemistry Laboratory, Geneva University Hospitals, Geneva, Switzerland4: CNRS, UMR 7177. Institut de Chime de Strasbourg, Strasbourg, France5: Universit Joseph Fourier, Grenoble FranceCorrespon
3、dence : Thierry Rabilloud, iRTSV/LBBSI, UMR CNRS 5092,CEA-Grenoble, 17 rue des martyrs, F-38054 GRENOBLE CEDEX 9Tel (33)-4-38-78-32-12Fax (33)-4-38-78-44-99e-mail: Thierry.Rabilloud cea.fr Abstract: Proteomics can be defined as the large-scale analysis of proteins. Due to the complexity of biologica
4、l systems, it is required to concatenate various separation techniques prior to mass spectrometry. These techniques, dealing with proteins or peptides, can rely on chromatography or electrophoresis. In this review, the electrophoretic techniques are under scrutiny. Their principles are recalled, and
5、 their applications for peptide and protein separations are presented and critically discussed. In addition, the features that are specific to gel electrophoresis and that interplay with mass spectrometry( i.e., protein detection after electrophoresis, and the process leading from a gel piece to a s
6、olution of peptides) are also discussed. Keywords: electrophoresis, two-dimensional electrophoresis, isoelectric focusing, immobilized pH gradients, peptides, proteins, proteomics. Table of contentsI. IntroductionII. The principles at playIII. How to use electrophoresis in a proteomics strategyIII.A
7、. Electrophoretic separation of peptides.III.A.1. Capillary IEFIII.A.2. Solution IEF III.A.3. IPG-IEFIII.A.4. Off-Gel III.A.5. A new dimension for data validationIII.B. Electrophoretic separation of proteins. III.B.1. Electrophoretic separations of native proteinsIII.B.2. Electrophoretic separations
8、 of denatured proteins-rationaleIII.B.3. The implementation of denaturing protein electrophoresis in proteomicsIII.B.4. The workhorse of protein separation in proteomics: SDS gel electrophoresisIII.B.5. Classical high-resolution 2D electrophoresis: the source of proteomicsIII.B.6. Specialized, IEF-f
9、ree, 2D electrophoretic systemsIV. From gels to peptides: additional features of electrophoresis in a proteomics strategyIV.A. Detection of proteins after gel electrophoresisIV.B. Production of peptides from gel-separated proteinsV. Concluding remarksVI. ReferencesI. IntroductionAmong the variety of
10、 available spectroscopic techniques, the combination of speed, sensitivity, and accuracy makes mass spectrometry an obvious choice in proteomics strategies; i.e., strategies that aim at the wide-scale characterization of proteins and protein variants in biological samples.However, mass spectrometry
11、alone cannot solve this analytical problem of the wide-scale characterization of proteins, for several major reasons. First of all, biological samples often contain several hundreds to several tens of thousands of different protein forms. The word of “protein forms” encompass, of course, gene produc
12、ts with all of their post-translational modifications (PTM); e.g., glycosylation, phosphorylation, or protein cleavage. On top of the diversity problem, the dynamic range of protein presence ( i.e., the mass ratio between the rarest protein form and the most abundant ones) is often far beyond the qu
13、antitative dynamic range of a mass spectrometer. For example, this dynamic range covers 4 orders of magnitude in prokaryotic samples, 6 in eukaryotic cells (Lu et al., 2007), and 12 in complex biological fluids such as plasma (Anderson and Anderson, 2002). Still going on this trend of complexity, th
14、e analyte itself (i.e, the protein) is very complex, so that even the most precise mass measurement of a complete, modified protein often does not give an unequivocal answer, in the sense that several modified proteins can fit within the same mass measurement window.Last but not least, it is quite f
15、requent in biology to have to address quantitation problems. A good example is provided by blood tests, in which normal and pathological values are defined, with the normal values frequently not zero. Thus, a quantitative approach is often needed, and this goal is not always easy to implement for ma
16、ss spectrometry. However, the discussion of the quantitation issues in mass spectrometry for proteomics is clearly outside the scope of this review. Because of the complexity of the analytes, the mass spectrometric measurement is not carried out on intact proteins, but on smaller peptides that are p
17、roduced from the proteins by a controlled proteolytic process. The multiple measurements made on peptide masses, either on a few peptides by MS/MS or on a combination of peptides that arise from the same protein (e.g., for peptide mass fingerprinting), allow an unequivocal characterization of the pr
18、oteins of interest and sometimes of some post-translational modifications.Because of the complexity of biological samples, it is absolutely necessary to separate (and sometimes to quantify) the analytes prior to their measurement with mass spectrometry, so that the complexity of what is introduced i
19、nto the mass spectrometer is compatible with the performance of the instrument in the chosen measurement mode. This separation can be made on the proteins of the sample or on the peptides that arise from protein digestion (or on both). Among the various separation modes available for proteins and pe
20、ptides, chromatography and electrophoresis are almost exclusively used at the current time. Thus the purpose of this review is to provide to the reader a critical review of the input of electrophoretic techniques in a proteomics strategy. II. The principles at playBy definition, electrophoresis cons
21、ists of the separation of analytes (here peptides or proteins) as ions that are driven differently (in order to achieve separation) under an electric field. Two modes of electrophoresis are mostly used for peptide and protein separation; zone electrophoresis and isoelectric focusing. In zone electro
22、phoresis, a speed race is made in the separation medium, usually at constant pH, and the speed of each analyte is dictated by its charge, which drives it under the electric field, and by the friction forces, which slow it down. In order to minimize the impact of diffusion, which always tends to broa
23、den the peaks of analytes, and also to minimize the impact of the volume of the initial sample on the final resolution, a special trick, called discontinuous electrophoresis, is used (Davis, 1964), schematized on figure 1. In this system, a composite separation medium with two phases is used, (figur
24、e 1, left panel). The sample is loaded on top of the first phase, called the concentration phase, where an isotachophoresis is carried out. Isotachophoresis (same speed electrophoresis) uses the rules of transport of ions in an electric field to build what is called a moving boundary, in which the i
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