Limitations of Two-Dimensional Electrophoresis. Figure 1 gives the general work flow in proteomics and Table 1 addresses their strengths and limitations. Two-dimensional electrophoresis (2DE) was developed two decades before the term proteomics was coined [66, 67] . The 2DE entails the separation of complex protein mixtures by molecular charge in the first dimension and by mass in the second dimension. 2DE analysis provides several types of information about the hundreds of proteins investigated simultaneously, including molecular weight, pI and quantity, as well as possible posttranslational modifications. 2DE is extensively used but mostly for qualitative experiments and this method falls short in its reproducibility, inability to detect low abundant and hydrophobic proteins, low sensitivity in identifying proteins with pH values too low (pH < 3) or too high (pH > 10) and molecular masses too small (Mr < 10 kD) or too large (Mr > 150 kD) [2] [3] [4] [5] . Poor separations of basic proteins due to "streaking" of spots and membrane proteins resolution [68] are limiting factors in 2DE. However, 2DE is the only technique that can be routinely applied for parallel quantitative expression profiling of complex protein mixtures such as whole cell and tissue lysates [69] and most widely used method for efficiently separating proteins, their variants and modifications (up to 15000 proteins). There are two ways to study posttranslational modifications by means of 2DE. First, posttranslational modifications that alter the molecular weight and or pI of a protein are reflected in a shift in location of the corresponding protein spot on the proteomic pattern. Second, in combination with Western blotting, antibodies specific for posttranslational modifications can reveal spots on 2DE patterns containing proteins with these modifications [70] . Protein extraction and solubilization are key steps for proteomic analysis using 2DE, highly hydrophobic proteins tend to precipitate during isoelectro focusing (IEF), low copy number and the insolubility of transmembrane proteins renders quantitative analysis of these peptides and polypeptides are very challenging [71] . In order to enhance protein extraction and solubilization, different treatments and conditions are necessary to efficiently solubilise different types of protein extracts [72, 73] . The major challenge for protein visualization in 2DE is the compatibility of sensitive protein staining methods with mass spectrometric analysis. Therefore, several fluorescent staining methods have been developed for the visualization of 2DE patterns, including sypro stainings and Cy-dyes [74] . Although sypro ruby [75] and silver staining [76, 77] have a comparable sensitivity, sypro ruby staining allows much higher reproducibility, a significantly wider dynamic range and less false-positive staining. In addition, sypro ruby allows for the detection of lipoproteins, glycoproteins, metalloproteins, calcium-binding proteins, fibrillar proteins, and low molecular weight proteins that are less "stainable" using other methods. Finally, a large number of protein spots on 2DE patterns contain several proteins with a similar pI. A pH gradient with a narrow range allows zooming into different proteins with the same molecular weight. Increased separation distance 40 × 40 cm gels using CA-IEF [78] could increase the proteome coverage up to 5000 proteins. Use of overlapping narrow range IPGs "Zoom" gels and increase in separation area could yield better membrane protein separation [79] . This technology, however, is biased against certain classes of proteins including low abundance and hydrophobic proteins.