Separation of Proteins

Prior to the analysis of protein expression and abundance levels, proteins first have to be isolated into a "purified" state. Although there are a variety of chromatographic procedures for achieving this, two-dimensional (2D) gel elec-trophoretic separation has been the method of choice in the recent past. However, other new methodologies are now emerging, each methodology having complimentary strengths and weaknesses:

1. Analysis of comparative expression—once separated, it is then necessary to carry out some form of analysis to assess the relative abundance of the proteins present.

2. Identification of protein species—once a set of proteins showing differences in abundance between two or more states have been identified, digestion of the proteins to peptides and further analysis using mass spectrometric methodology can be used to determine their identities.

3. Confirmatory experiments—when a protein has been shown to be important in a given process by the above analysis, it may be necessary to perform further experiments to confirm its implied function or involvement in the process.

For proteomes that encompass the protein content of a given cell or tissue type, or that of a whole organism, there are two main methods that are first used to resolve the protein mixture, and then to visualize the individual components in such a way that their relative abundances can be quantified. The first method utilizes 2D polyacrylamide gel electrophoresis (2D-PAGE) followed by a variety of in-gel staining methods, whereas the second—more recent— technology couples liquid chromatographic separation to subsequent ultraviolet and/or mass spectrometric (MS) detection. This chapter focuses on 2D-PAGE

as the separation technique because this approach is better established and widely used in nonspecialist laboratories.

2D-PAGE has been routinely used over the past three decades to resolve and investigate several thousand proteins in a single sample. This has enabled identification of the major proteins in a tissue or subcellular fraction by MS methods. In addition, 2D-PAGE has been used to compare relative abundances of proteins in related samples, such as those from altered environments or from mutant and wild-type, thus allowing the response of classes of proteins to be determined. Problems with matching of spots from one gel to another, running variations, and the dynamic range of stains have limited quantitative studies. Visualization of spots on 2D-PAGE gels has traditionally involved silver staining, as it is more sensitive than conventional Coomassie staining methods. Silver staining is unsuitable for quantitative analysis; however, as it has a limited dynamic range, and the most sensitive of silver staining methods are also incompatible with protein identification methods based on mass spectrom-etry. More recently, the Sypro postelectrophoretic fluorescent stains (Invitrogen, Carlsbad, CA) have emerged as alternatives, offering a better dynamic range, and ease of use (3). Difference gel electrophoresis (DIGE), first described some time ago (4), circumvents issues with gel-to-gel variation and limited dynamic range and allows more accurate and sensitive quantitative proteomics studies. This technique relies on pre-electrophoretic labeling of samples with one of three spectrally resolvable fluorescent CyDyes (Cy2, Cy3, and Cy5), allowing multiplexing of samples into the same gel. There are currently two types of CyDye labeling chemistries available from GE Healthcare. The most established is the "minimal labeling" method. Here, the CyDyes are supplied as N-hydroxy succinimidyl esters, which react with primary amino groups. The stoichiometry of labeling is such that about 2% of available lysine residues are labeled. The CyDyes carry a positive charge and hence a labeling event does not alter the isoelectric point (p/) of the protein. In the second chemistry, uncharged CyDyes are supplied with a thiol-reactive maleimide group. These "saturation" dyes are utilized in such a way to bring about labeling of every cysteine residue within the protein. The saturation labeling is much more sensitive, as more fluorophor is introduced into each protein species (5). The use of these saturation dyes is not well established; therefore, this chapter focuses only on the minimal labeling of lysine residues.

For multiple gel studies such as a time course, samples can be labeled with either Cy3 or Cy5 minimal dyes, whereas Cy2 minimal dye is reserved for an internal standard sample. Up to three distinct labeled samples are run in one gel and viewed individually by scanning the gel at different wavelengths. Variation in spot volumes owing to gel-specific experimental factors—for example,

Wild-type sample

Wild-type sample

Internal standard

Gel B

GeIC

Wild-type sample

Wild-type sample

Internal standard

Gel B

GeIC

Fig. 1. Schematic diagram of a two-dimensional polyacrylamide gel with three spectrally resolvable samples resulting from the labeling with CyDyes, which highlights the importance of the internal standard in accounting for experimental variation. For the spot circled, if an internal standard were not included when comparing gel A to gel B it would be concluded that the protein expression had increased in the mutant samples. When using the internal standard to account for running success, it would be concluded that protein expression had actually decreased. Similarly, if gels A and C were compared without the internal standard it would be concluded that the protein was absent in the mutant samples where in fact the protein has not resolved on gel C. The inclusion of internal standard in the generation of standardized abundances can therefore take into account the experimental variation, allowing reproducible quantitation.

Fig. 1. Schematic diagram of a two-dimensional polyacrylamide gel with three spectrally resolvable samples resulting from the labeling with CyDyes, which highlights the importance of the internal standard in accounting for experimental variation. For the spot circled, if an internal standard were not included when comparing gel A to gel B it would be concluded that the protein expression had increased in the mutant samples. When using the internal standard to account for running success, it would be concluded that protein expression had actually decreased. Similarly, if gels A and C were compared without the internal standard it would be concluded that the protein was absent in the mutant samples where in fact the protein has not resolved on gel C. The inclusion of internal standard in the generation of standardized abundances can therefore take into account the experimental variation, allowing reproducible quantitation.

protein loss during sample entry into the immobilized pH gradient strip—will be the same for each sample within a single gel. Consequently, the relative amount of a protein in a gel in one sample compared with another will be unaffected (Fig. 1). In a multiple-gel experiment, the Cy2 is used to label a pooled sample consisting of equal amounts of each of the samples to be compared, and acts as an internal standard. This ensures that all proteins present in the samples are represented, allowing both inter- and intragel matching. The spot volumes are normalized for dye discrepancy, arising from differences in laser intensities, fluorescence, and filter transmittance, using a method based on the assumption that the majority of protein spots have not changed in expression level (6). The spot volumes from the labeled samples are compared with the internal standard giving standardized abundances, which allows the variation in spot running success to be taken into consideration.

For the analysis, software developed for the DIGE system, such as DeCyder™ (GE Healthcare, Uppsala, Sweden) is typically used. This software has a codetection algorithm that simultaneously detects labeled protein spots from images that arise from the same gel and increases accuracy in the quantification of standardized abundance (6). The standardized abundances can then be compared across groups to detect changes in protein expression (see Fig. 2 for a sample time course profile obtained from a multiple-gel DIGE experiment). The technical improvements in this field have made possible more complex experimental designs in proteomics expression studies, such as a time course or a moving window approach. Given that proteins are separated by both pi and molecular weight (MW), certain posttranslational modifications that result in a change in either of these parameters are visible. Successive phoshorylation events, for example, lead to a "charge train" of spots as the phosphorylation event decreases the pi of the protein. Consequently, DIGE has the potential to identify changes that arise not only from changes in protein levels but also from posttranslation modifications (see Fig. 3 for an example). To date, the DIGE technology has been used with great success to study a variety of systems, allowing the detection of more subtle changes in protein expression than conventional methods in which separate samples are loaded onto each gel (7-12).

Regardless of the benefits or DIGE, the 2D-PAGE process itself has some limitations. For global expression analysis, every protein should be resolved as a discrete detectable spot; however, the following groups of proteins are often poorly represented: those with extreme pi or MW; hydrophobic proteins; lower abundance proteins. It has been calculated that somewhere in the region of 90% of the total protein of a typical cell is made up of only 10% of the 10,000 to 20,000 different species, and hence many low-abundance proteins may not be detectable (13). Improvements to the technique are ongoing, such as increasing resolution of protein species by the use of narrow-range immobilized pH gradient (IPG) strips. Moreover, prefractionation of samples has been demonstrated and greatly improves the chance of identification and assignment of function to low-abundance species (14,15).

Fig. 2. Examples of changes in protein expression as seen as changes in the standardized log abundance obtained for a time-course multiple-gel difference gel electro-phoresis experiment.

The stages required for the DIGE approach in the identification of proteins with expression changes are shown in Fig. 4 as a flow diagram and are outlined in more detail in Heading 3.

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