Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 51
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Arecent example, looking at 2DE electrophoresis of Saccharomyces cerevisiae, revealed an increase in the number of protein spots detected in a particular samplewhen narrow-range pH gradient strips were used. Using a pH 3–10 gradient, 755protein spots were detected in the second dimension which rose to a total of 2286protein spots being detected in the same sample across a number of 2DE gelswhen a selection of narrow-range pH gradients were employed in the first dimen-17717811 Proteomics, a Step Beyond GenomicsFig.
11.3 a, b Zoom gels: increasing proteomic coverage using narrow-range pH gradients. Fig. 11.3a shows human heart (ventricle)proteins which were initially separated in thefirst dimension using an 18 cm non-linear pH3–10 immobilised pH gradient (IPG). In orderto separate this sample still further, very narrow-range pH gradients were used (e.g. pH4–7L, 6-9L, 4–5L, 5–6L, 3.5–4.5L, 5.5–6.7L).The second dimension for all gels was a21 cm 12% SDS-PAGE (sodium dodecylsulphate polyacrylamide gel electrophoresis) gel.Proteins were detected by silver staining.
Theadvantages of using this approach are illustrated in Fig. 11.3 b. One protein spot is detected by silver staining on a 2DE gel afterusing a pH 3–10 IPG in the first dimension.Analysis by MS (mass spectrometry) identifiedthis spot as either enoyl-CoA-hydratase (EH) orHSP27 (Fig. 11.3 bi). If the same sample is separated further in the first dimension using anarrower pH 4–7 IPG, this same spot nowbegins to separate out into two protein spots.Spot 1 was identified by MS as EH and spot 2as HSP27 (Fig. 11.3bii).
Increasing the resolution even further by employing a pH 5.5–6.7IPG in the first dimension allows three proteinspots to now be detected in this region by silverstaining. Subsequent MS analysis identifiedspot 1 and 2 as EH and spot 3 as HSP27(Fig. 11.3biii). (Figures 11.3a and 11.3b takenfrom Westbrook et al, 2001, 22 2865–2871).11.4 Protein Detection/VisualisationFig.
11.3 bsion [20]. An important additional advantage of narrow range or so-called zoomIPG’s is that they can tolerate higher protein loading for micro-preparative purposes. By generating 2DE gels using IPG strips with overlapping pH ranges it ispossible to view the gels side-by-side to form the equivalent of proteomic ‘contigs’[21]. As well as narrow pH gradients, very basic gradients up to pH 12 are nowavailable, and are useful for separating very basic cellular components e.g. nuclearand ribosomal proteins [22].An additional/alternative approach to increasing proteomic coverage, termedsubproteomics, is subcellular fractionation. This is based upon sample prefractionation into specific cellular compartments to determine protein location.
Combining this with immobilized pH gradients spanning single pH units can be a powerful tool to use to visualize poorly abundant proteins within a cellular system [23].11.4Protein Detection/VisualisationIn order to visualize proteins after electrophoresis they have to be detected at highsensitivity. Therefore the stain used should ideally possess a high dynamic range(i.e. it can detect proteins over a wide range of concentrations and linearity to allow rigorous quantitative analysis). Also, it is an advantage if the staining methodused is compatible with subsequent protein identification by mass spectrometry.Visualisation of proteins fixed in a gel is usually achieved by staining the wholegel.
Traditionally protein staining following gel electrophoresis was performedusing Coomassie Brilliant Blue (CBB), however this has very limited sensitivity.17918011 Proteomics, a Step Beyond GenomicsWhile increased sensitivity can be achieved using CBB in a colloidal form [24],a preferred alternative is silver staining of polyacrylamide gels. This was first introduced in 1979 by Switzer et al. [25] and quickly became very popular due to itshigh sensitivity (approximately 0.1 ng protein per spot, some 100 times more sensitive than Coomassie blue). However, silver staining is not without its own problems.
Using this method as a quantitative procedure can be challenging becauseit is known to be non-stoichiometric and prone to saturation and negative staining effects, where regions of very high protein concentration do not stain and appear as ‘holes’ in the pattern of stained spots. Not only does silver staining pose aproblem when trying to quantify differential protein expression but it can also interfere with subsequent identification by mass spectrometry (MS). The reason forthis is the inclusion of gluteraldehyde in the sensitization step of silver staining.Gluteraldehyde reacts with the amino groups of proteins, both e-amino (lysineside chain) and a-amino, causing extensive cross-linking of proteins.
To achieveoptimal MS analysis of silver stained proteins, gluteraldehyde must be eliminatedfrom the sensitization step [26–28].Detection methods based on the use of fluorescent compounds are claimed tohave a much larger linear dynamic range, and hence sensitivity, than silver staining. Over recent years we have seen the introduction of fluorescent post-electrophoretic stains such as Sypro Ruby and Sypro Orange which are said to offer improved sensitivity, higher dynamic range and easy handling [29]. Images of fluorescently stained gels can be easily visualized/captured using UV tables and multiwavelength fluorescent scanners.Extensive studies have been carried out to evaluate the sensitivity of these stainsin comparison to silver staining [30, 31].
In our laboratory we have comparedthree commercially available fluorescent stains, SYPRO Ruby, SYPRO Orange andSYPRO Red (Molecular Probes, Eugene, OR, USA), and compared their sensitivityand dynamic range on 2DE gels with silver staining [32]. We found that SYPRORuby had a similar sensitivity to silver staining, but SYPRO Orange and SYPRORed were considerably less sensitive, requiring respectively four and eight timesmore protein to be loaded to achieve equivalent 2DE protein profiles. Not unexpectedly, silver staining provided pour linearity even for the less abundant proteins where saturation was not a problem. In contrast the SYPRO dyes providedmuch greater linearity over their extended dynamic range [32].Recently much effort has been put in to investigate the compatibility of fluorescent stains, in particular Sypro Ruby, with mass spectrometry.
The general findingis that in comparison with conventional silver staining, Sypro Ruby demonstratesa broad linear dynamic range and enhanced recovery of peptides from in-gel digests for matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)mass spectrometry [30, 33]. In addition to increased sensitivity, ease of use is another advantage to the fluorescent stain and the user, since staining a gel usingSypro Ruby follows a much simpler and far less labour intensive protocol than silver staining.Whilst fluorescent post-electrophoretic stains have increased the sensitivity ofdetection and quantitation of proteins on 2-D gels, many pairs of gels are still re-11.4 Protein Detection/Visualisationquired in order to establish biologically statistically significant differences betweenfor example a control and disease state. However, the development of fluorescent2DE differential gel electrophoresis (DIGE) by Unlu et al. [34] has now enabled usto be able to detect and quantitate differences between experimentally paired samples resolved on the same 2DE gel.
DIGE uses two mass- and charge-matched Nhydroxy succinimidyl ester derivatives of the fluorescent cyanine dyes Cy3 andCy5, which possess distinct excitation and emission spectra. Cy3 and Cy5 covalently bind to lysine residues. Two samples that are to be compared are both labeled with each of the dyes. Once both samples are labeled, equivalent aliquots ofeach are mixed and the resulting mixture of both samples is then subjected to2DE (Fig. 11.4). The resulting gel is fluorescently imaged twice (once for the Cy3dye and once for the Cy5 dye) and then both images are superimposed.
The labeling process for DIGE takes only 45 minutes which is much faster than stainingFig. 11.4 DIGE (differential gel electrophoresis) analysis. A flow diagram illustrating theuse of Cy3, Cy5 and Cy2 dyes in the analysisof control versus diseased samples. One protein sample is labeled with Cy3 (e.g. controlsample), whilst the other is labeled with Cy5(e.g. diseased/experimental sample). After thelabeling reaction is terminated, equalamounts of each labeled sample are combined.
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