|X-ray diffraction with the D8 FOCUS can identify and quantify the different phases inside a powder sample. The same data also provides information on the crystallite size and the microstrain.||
One of the greatest challenges of proteome analysis is the reproducible separation of complex protein mixtures while retaining both qualitative and quantitative relationships. Many combinations of techniques can be used to separate and analyze proteins, but two-dimensional (2-D) electrophoresis is uniquely powerful in its ability to separate hundreds to thousands of products simultaneously (Choe and Lee 2000)2. This technique uses two different electrophoretic separations, isoelectric focusing (IEF) and SDS-PAGE, to separate proteins according to their isoelectric point (pI) and molecular weight.
The identities of individual protein spots from the gel can then be identified by mass spectrometry (MS) of their tryptic peptides. Together with computerassisted image evaluation systems for comprehensive qualitative and quantitative examination of proteomes, proteome analysis also allows cataloguing and comparison of data among groups of researchers.
Protein electrophoresis is the movement of proteins within an electric field. Protein electrophoresis can be used for a variety of applications such as purifying proteins, assessing protein purity (for example, at various stages during a chromatographic separation), gathering data on the regulation of protein expression, or determining protein size, isoelectric point (pI), and enzymatic activity. In fact, a significant number of techniques including gel electrophoresis, isoelectric focusing (IEF), electrophoretic transfer (blotting), and two-dimensional (2-D) electrophoresis can be grouped under the term “protein electrophoresis” (Rabilloud 2010)3. Factors affecting protein electrophoresis include the strength of the electric field, the temperature of the system, the pH, ion type, and concentration of the buffer as well as the size, shape, and charge of the proteins (Garfin 1990)4.
Effective sample preparation is key for the success of the experiment. The sample dictates the type of extraction technique used, and the solubility, charge, and pI of the proteins of interest affect the method of solubilization. Sample preparation contributes significantly to the overall reproducibility and accuracy of protein expression analysis (Link 19995, Rabilloud 19996, Molloy 20007). Without proper sample preparation, proteins may not separate from one another or may not be represented in the 2-D pattern. Since protein types and sample origins show great diversity, there is no universal sample preparation method. In addition, some proteins simply cannot be solubilized under conditions compatible with IEF. Sample preparation procedures must be optimized empirically and tailored to each sample type and experimental goal.
Proteins separated in gels are usually not visible to the naked eye and must, therefore, be either stained or labeled for visualization. Several factors determine the best choice of staining method, including desired sensitivity, linear range, ease of use, expense, and the type of imaging equipment available.
The ability to collect data in digital form is one of themajor factors that make 2-D gels a practical meansof collecting proteome information. It allows theunbiased comparison of samples and gels, transfer ofinformation among research groups, and cataloguingof data. Many types of imaging devices interface withsoftware designed specifically to collect, interpret,and compare proteomics data.
- Wilkins MR et al. (1996). Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. Biotechnol Genet Eng Rev 13, 19–50.
- Choe LH and Lee KL (2000). A comparison of three commercially available isoelectric focusing units for proteome analysis: the multiphor, the IPGphor and the PROTEAN IEC cell. Electrophoresis 21, 993–1000.
- Rabilloud T (2010). Variations on a theme: changes to electrophoretic separations that can make a difference. J Proteomics 73, 1562–1572.
- Garfin DE (1990). One-dimensional gel electrophoresis. Methods Enzymol 182, 425–441.
- Link AJ ed. (1999). 2-D Proteome Analysis Protocols (New Jersey: Humana Press).
- Rabilloud T (1999). Solubilization of proteins in 2–D electrophoresis. An outline. Methods Mol Biol 112, 9-19.
- Molloy MP (2000). Two – dimensional electrophoresis of membrane proteins using immobilized pH gradients. Anal Biochem 280, 1 – 10.