Protein separation using gel based techniques

The modern approach of proteomics consists of two basic elements: the separation of the complex mixture of proteins, and identification of the individual proteins. High resolution separations are the most important feature in separation sciences, in order to be able to analyse complex samples especially in the biological samples. Electrophoretic separations of proteins are no exception to this rule, with the additional difficulty that proteins are very complex substances that have a strong tendency to precipitate (Graves & Haystead, 2002). Two high- performance electrophoretic separations of proteins were available at the very beginning of the 70s, the first one is electrophoresis of proteins in the presence of SDS, as described by (Laemmli, 1970), a technique that instantly became very popular, and the second one is denaturing isoelectric focusing, as described by (Gronow & Griffith, 1971). As these two techniques used completely independent separation parameters (molecular mass and isoelectric point, respectively) it is not surprising that it was soon tried to couple them. The first successful, detailed study, utilising these two different parameters was reported by (O'Faffel, 1975). Subsequently, this technique got its famous name the two-dimensional polyacrylamide gel electrophoresis 2D-PAGE (Rabilloud & Lelong, 2011).

Some complex biological sample may contain too many proteins or peptides for the mass spectrometer to detect them all at once, so separation techniques can be utilised to reduce the complexity of an analyte proteome. By far, the most common technique for the analysis of either a subset of proteins or all resolvable proteins in a cell is gel electrophoresis including either 1D sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE or two-dimensional polyacrylamide gel electrophoresis 2D-PAGE. When using SDS-PAGE, the proteins are both solubilised and given a charge by the detergent SDS and further separated according to their molecular weight. Negatively charged proteins traps in the polyacrylamide matrix (gel) as they travel towards a positive electrode (anode), facilitated by an electric field applied across the gel. The ionic detergent SDS is added to the proteins where it binds and denatures the proteins, distributing a negative charge evenly across the whole protein. SDS-PAGE is simple to perform, reproducible and can separate proteins with molecular weights between 10 and 300 kDa. For many applications SDS-PAGE is the method of choice, but due to its limited resolving power it is only suitable for separation of mixtures after some form of purification (John, 2002). Reducing SDS-PAGE is usually performed, which includes the addition of dithiothreitol (DTT)

35 to the sample, this denatures the proteins further by breaking any disulphide bonds and disrupting any tertiary protein folding and quaternary structure, giving an improved protein

separation (Claudia et al., 2012).

When separation of more complex samples is required, such as total cell lysates, 2D- PAGE is the method of choice. 2D-PAGE is an extension step of the 1D separation with the addition of isoelectric focusing (IEF) before SDS-PAGE separation. IEF of proteins is performed along an acrylamide gel strip with an immobilised pH gradient, where proteins are separated based on their charge in a specific buffer and their differing isoelectric points, which is the point on the pH gradient where each protein has no net charge. Proteins migrate along the gel with the electric field to their isoelectric point. This immobilised pH gel is then added to the top of a large polyacrylamide gel and SDS-PAGE is performed (Figure 2.10). The combination

of these two methods achieves resolution far exceeding that of SDS-PAGE alone (Rogowska et

al., 2013).

Figure 2.10: 2D Electrophoresis. Protein spot result from two separations: first by pI (IEF) and

36 Isoelectric focusing became more reproducible with the development of immobilised pH gradients (IPG) and Immobiline™, eliminating the possibility of the pH gradient drift and brought superior resolution and reproducibility to first dimension IEF. The primary application of 2D-PAGE is expression the profile of complex protein mixtures extracted from cells, tissues, or other biological samples, when the comparison of the resulting images of two samples is possible, and that gives both quantitative and qualitative information. Another application of 2D-PAGE is monitoring post-translational modifications, which is possible because most of the modifications would alter the charge and the molecular weight of the protein. Currently there are many software packages designed to detect, match and calculate the volumes of spots between gels, this enables quantitative and statistical analyses. For instance, BioNumerics 2D, Delta 2D, ImageMaster 2D, and many others. Protein spots of interest can be excised from the 2D-PAGE, digested, and the peptides subjected to mass spectrometry (MS) to identify the parent protein (Yerlekar & Dudhe, 2014).

With all advantages and features of 2D-PAGE based proteomics, there are still a number of limitations when using this approach. Major shortcomings of 2D-PAGE include the poor representation of basic and membrane proteins (Due to poor solubility in non-ionic lysis buffers and inefficient transfer from the isoelectric focusing strip to the SDS-PAGE gel), limited dynamic range and the potential for “hidden” proteins, 2D-PAGE spots may be made up of more than one protein (two or more may have similar isoelectric point and molecular weights), as well as tendency towards high abundance proteins (isoelectric point of 4-7), and finally the 2D- PAGE is time consuming to run and manually intensive. These disadvantages of 2D-PAGE demonstrate that the choice of method depends largely on the type of sample and its complexity (Wittmann-Liebold, Graack, & Pohl, 2006).