Strategies for Protein Separation

Abstract

The proteome of a cell or tissue depends on cellular and environmental conditions, showing a dynamic system subject to large variations. To study these large changes of variability and quantity, proteomics has emerged, providing techniques dedicated to global characterization of all proteins simultaneously. The expectation is that this information will produce new insights into the biological function of proteins in different physiological states of a cell or tissue. The proteome has a dynamic and complex nature that is the result of many posttranslational modifications, molecular interactions, and a variety of proteins arising from alternative mRNA splicing. With this in mind, the number of modified and unmodified proteins found in any biological system is much bigger than the number of genes (Anderson et al., 2004), which is why mRNA expression may not correlate with protein content (Rogers et. al, 2008). In addition, not all proteins are expressed in the same or similar level in the proteome. For example, the enzyme Rubisco comprises 3050% of leaf proteome (Feller et al., 2008), which is a big issue in the proteomic assessment of low-abundance proteins. In fact, the majority of proteins are in the low-abundance level. To overcome these challenges, the proteome must be fractionated for effective detection and quantification by mass spectrometry (MS). Consequently, the analysis of proteins on the large or small scale is dependent on separation methods. As the ultimate goal in proteomics is to resolve all individual proteins in the cell, although it is quite difficult to find a separation method that could accommodate the diversity of proteins equally, protein separation methods directly affect the achievement of reliable results. Such methods are based on the physical or chemical properties of different proteins, such as their mass or net charge. The combination of sequential methods exploiting different properties can provide highresolution analysis of very complex protein mixtures. Then, current analytical strategies can reach different levels of resolution depending on the platform used. Two-dimensional gel electrophoresis (2DGE) and multidimensional liquid chromatography (MDLC) are the two methods that dominate the separation steps in proteomics. The differences of each strategy are basically related to sensitivity, automation, and high-throughput possibilities. In this chapter, the limitations and principles of these techniques will be discussed.