Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 50
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van Eyk, Michael J. DunnCopyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 3-527-30596-317411 Proteomics, a Step Beyond GenomicsWhilst there are many new technologies that have been developed to investigatechanges in protein expression and PTM’s such as LC-MS/MS, discussed in Chapter 11 and ICATTM, discussed in Chapter 12, here we will focus on protein separation, visualization, and quantitation using the traditional 2DE approach.11.2Protein Solubilization2DE is a powerful technique which can be used to analyse the proteome and hasthe advantage of being able to resolve and reveal post-translational modificationsof proteins, as these generally result in changes in protein charge and/or mass.
Ithas become the method of choice for the analysis of protein expression in complex biological systems such as whole cells, tissues and organisms. In order to exploit this technique to the full, sample preparation is of paramount importance.Maximising protein solubilization is key to resolving any proteome.
To this end,the correct choice of solubilization buffer is vital. Samples are generally preparedusing denaturing lysis buffers containing a high urea concentration (7–9 M) withthe addition of a non-ionic (e.g. NP-40) or zwitterionic (e.g. CHAPS) detergentand a reducing agent (e.g. DTT). These conditions are intended to break all thenon-covalent interactions between the sample proteins [8]. Preventing any artifactual or chemical modifications once sample proteins are solubilized is imperative.Solubilization buffer should maintain all extracted polypeptides in their intactstate (amino acid composition and PTM’s), without introducing further artifactualmodifications, prior to solubilization. Therefore all enzymes that are able to modify proteins, such as proteases, must be quickly and irreversibly inactivated.
It isimportant to understand that any one solubilization buffer, e.g. the ‘standard’ buffer described above, is not suitable for all proteins. Currently the major challengein proteomics is the development of appropriate methods for the solubilization ofparticular classes of proteins such as hydrophobic membrane and membrane-associated proteins. Variations in solubilization buffer constituents using newly developed detergents, e.g. sulfobetaine, additional denaturing agents such as thiourea,and alternative reducing agents, e.g. trubutyl phosphine, can help to improve protein solubilization and hence the concentration of extracted protein for certainsample types [9, 10].
The choice of solubilisation buffer must be optimized foreach sample type to be analysed by 2DE.11.3Protein SeparationProtein separation by 2DE involves two steps. The first step or dimension subjectssolubilized proteins to isoelectric focusing (IEF) under denaturing conditions. Onthe application of a current, the charged polypeptide subunits migrate along apolyacrylamide gel strip that contains a pH gradient of an appropriate range, until11.3 Protein Separationthey reach the pH at which their net charge is zero (isoelectric focusing point orpI). The gel strip thus contains discrete protein bands along its length, separatedupon the basis of their charge.
This is combined with a second-dimension separation on sodium dodecyl sulfate (SDS) polyacrylamide gels where the gel strip isapplied to the edge of the second-dimension SDS gel and the focussed polypeptides migrate in an electric current into the second gel, proteins now separatingon the basis of their relative molecular mass. This orthogonal combination ofcharge separation (isoelectric point, pI) with size separation (relative molecularmass, Mr) results in the sample proteins being distributed across the two-dimensional gel profile (Fig.
11.1). Resulting protein spot patterns are conventionally orientated with acidic isoelectric points to the left of the gel and low molecularweight proteins at the bottom of the gel (Fig. 11.1).To be effective and accurate 2DE must produce highly reproducible protein separations and have high resolution. This has been achieved in recent years by theuse of immobilized pH gradients (IPG) (Amersham Biosciences) [11].
Previouslysynthetic carrier ampholytes (SCA) were used to generate the pH gradients required for IEF. Electroendosmotic flow of water (migration of H3O+ towards theFig. 11.1 A two-dimensional electrophoresis(2DE) separation of 80 lg of heart (ventricle)proteins. The first dimension comprised an18 cm non-linear pH 3–10 immobilised pHgradient (IPG) subjected to isoelectric focusing. The second dimension was a 21 cm 12%SDS-PAGE (sodium dodecylsulphate polyacrylamide gel electrophoresis) gel. Proteins weredetected by silver staining.
The non-linear pHrange of the first-dimension IPG strip is indicated along the top of the gel, acidic pH tothe left. The apparent isoelectric points (pI)of the separated proteins can be estimatedfrom these values. The Mr (relative molecularmass) scale can be used to estimate the molecular weights of the separated proteins.17517611 Proteomics, a Step Beyond Genomicscathode during electrophoresis) occurring during IEF results in migration of thesmall SCA molecules towards the cathode. This is known as cathodic drift and results in pH gradient instability and the loss of the more basic proteins from thegel. The application of IPG technology to 2DE overcame this problem.
The immobiline reagents consist of a series of eight acrylamide derivatives with the structure CH2=CH-CO-NH-R, where R contains either a carboxyl or tertiary aminogroup, giving a series of buffers with different pK values distributed throughoutthe pH 3 to 10 range. The appropriate IPG reagents can be added to the gel polymerization mixture according to published recipes. During the polymerizationprocess the buffering groups that form the pH gradient are covalently attached viavinyl bonds to the polyacrylamide backbone.
IPG generated in this way are immune to the effects of electroendosmosis. IEF separations are therefore extremelystable. As with anything new, implementing the IPG technology to 2DE separations proved problematic. However, these were overcome and now IPG IEF isthe current method of choice for the first dimension of 2DE [12]. IPG IEF is performed on individual gel strips, 3–5 mm wide, cast on a plastic support. A rangeof pre-cast IPG strips carrying various pH ranges are available commercially (IPGDryStrips, Amersham Biosciences; IPG Ready Strips, Bio-Rad Laboratories).
IEFis carried out generally using a horizontal flat-bed IEF system (e.g. IPGphor,Amersham Biosciences; Multiphor, Amersham Biosciences; Protean II XL Cell,Bio-Rad). Following steady-state IEF, strips are equilibrated and then applied tothe surface of either vertical or horizontal slab SDS-PAGE gels as described above[12]. Using this method, inter-laboratory studies of heart, barley and yeast proteinshave demonstrated that excellent reproducibility can be achieved both in proteinsmigrating to the same position on a gel and quantitative data [13, 14].Recently, improvements in protein separation in the first dimension have beenreported, where a combined IPGphor/Multiphor approach has been used [15].The main difference between the two systems is that in-gel rehydration using theIPGphor is carried out under low voltage overnight whereas with the Multiphor itis carried out using rehydration trays at room temperature.
An additional advantage, in theory, is that IEF can be performed much faster at higher voltages (up to8 kV) using the IPGphor. The advantages associated with both pieces of equipment when performing IEF are controversial. By applying a low voltage during ingel rehydration, improved protein entry, especially of high molecular weight proteins has been reported [16]. However, an increased number of detectable proteinspots in 2DE gels has been reported following IEF using the Multiphor comparedto the same samples subjected to IEF using the IPGphor [17].
By using the IPGphor for sample loading under a low voltage, then transferring strips to the Multiphor for focusing, an increased sample entry compared to using the Multiphoralone was visualized in the resulting gel pattern [15].Using standard format SDS-gels (20 ´ 20 cm) with 18 cm IPG strips it is possible to routinely separate 2000 proteins from whole-cell and tissue extracts. Resolution can be significantly enhanced (separation of 5000–10,000 proteins) usinglarge format (40 ´ 30 cm) 2DE gels [18].
However, gels of this size are very rarelyused due to the handling problems associated with such large gels. In contrast,11.3 Protein SeparationFig. 11.2 Small format (7 ´ 7 cm) 2DE gel separations of human heart (ventricle) proteins. Inthe first dimension proteins were separated by isoelectric focusing along a linear pH 3-10 gradient and the second dimension was a 12% SDS-PAGE (sodium dodecylsulphate polyacrylamidegel electrophoresis) gel. Proteins were detected by silver staining (A) 4 lg, (B) 6 lg protein loading.mini-gels (7 ´ 7 cm) can be run using 7 cm IPG strips. Whilst these gels will onlyseparate a few hundred proteins they can be very useful for rapid screening purposes or for studies where the amount of protein available is limited (Fig.
11.2).Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in thesecond dimension is performed using apparatus dedicated to running multiplelarge-format 2DE SDS-gels. Currently available commercial gel-tanks have the capacity to run batches of up to twelve gels at one time (e.g.
DALT 2 Ettan DALT,Amersham Biosciences; Protean Plus Dodeca Cell, Bio-Rad). The main advantagethat these gel-tanks have over older models is that gels can be run in only 5–6hours.Separation and resolution can be further maximised using IPG strips coveringa variety of pH ranges. Wide pH 3–10 gradients are used to give an overview ofthe protein profile of a sample. Narrow range IPG’s (e.g pH 4–7, 6–9, 4.0–5.0,4.5–5.5, 5.0–6.0, 5.5–6.7) are now available and have the capability of ‘pullingapart’ this protein profile and increasing the resolution, and thereby increasingproteomic coverage, in particular regions (Figs. 11.3 a & 3 b) [19]. Thus, the task ofresolving more and more proteins and increasing the amount of informationabout a particular proteome becomes easier but perhaps a little interminable.
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