Proteins high-resolution separation

Capillary zone electrophoresis, an up-to-date high resolution separation method useful for proteins and peptides, has been shown to be a useful method for determining electrophoretic mobilities and diffusion coefficients of proteins [3], Diffusion coefficients can be measured from peak widths of analyte bands. The validity of the method was demonstrated by measuring the diffusion coefficients for dansylated amino acids and myoglobin. 

We therefore sought to evaluate reproducibility of shotgun proteomics in studies of archival FFPE tissue. Because FFPE samples are more complex than non-cross-linked samples, we evaluated FFPE human liver for analytical reproducibility and confidence in protein assignments.20 This complexity strengthens the argument for using high-resolution separations to maximize analyte concentration and minimize matrix effects. In this case, we used transient capillary isotachophoresis/capillary zone electrophoresis (cITP/cZE) in place of IEF to help address this effect. cITP/cZE has a resolution superior even to cIEF (90% of identified peptides in 1 fraction, 95% in 2 fractions or less for cITP/cZE, vs. 75% and 80%, respectively, for cIEF). 

SDS polyacrylamide gel electrophoresis (SDS-PAGE) represents the most commonly used analytical technique in the assessment of final product purity (Figure 7.1). This technique is well established and easy to perform. It provides high-resolution separation of polypeptides on the basis of their molecular mass. Bands containing as little as 100 ng of protein can be visualized by staining the gel with dyes such as Coomassie blue. Subsequent gel analysis by scanning laser densitometry allows quantitative determination of the protein content of each band (thus allowing quantification of protein impurities in the product). 

The reproducible high-resolution separation of protein mixtures is the main purpose of proteome analysis. O FarelTs classic tube gel technique has limited reproducibility. It is often difficult to compare the protein profiles obtained using O FarelTs method in different laboratories. In some cases, the data obtained even in the same laboratory by different operators are not comparable. 

The figure in Box 3-C shows a high-resolution separation of the soluble proteins of E. coli. The investigator labeled the proteins with -containing amino acids. 

Each column type has its own place of use. Column variety is what gives HPLC its versatility. It really depends on your compound and application. Approximately 80% of all separations are done on 5-10-jUm reverse phase Ci8 silica columns. Much of this is tradition. Reverse phase columns offer high-resolution separations for a wide variety of compounds and can be run in aqueous mobile phases. Ion exchange separations require salt solutions for separations, and these are not compatible with mass spectrometers. Size separations have lower resolving power and longer run times, but may be the only way to separate proteins solutions that will irreversibly stick to reverse phase columns. Use small pore size separation columns to remove salt from effluent from other chromatography separations. Zirconium and polymeric column are newer and offer possibilities for unique separations. 

Isoelectric focusing can be combined with SDS-PAGE to obtain very high resolution separations in a procedure known as two-dimensional gel electrophoresis. The protein sample is first subjected to isoelectric focusing in a narrow strip of gel containing polyampholytes. This gel strip is then placed on top of an SDS-polyacrylamide gel and electrophoresed to produce a two-dimensional pattern of spots in which the proteins have been separated in the horizontal direction on the basis of their pi, and in the vertical direction on the basis of their mass (Fig. 4). The overall result is that proteins are separated both on the basis of their size and their... 

While all of these devices used normal zone electrophoresis separation techniques, isoelectric focusing (IEF) methods on microchips have also been interfaced with ESI-MS for high-resolution separations of proteins [37], Figure 5 shows the electropherogram and corresponding mass spectra generated by this device. For on-chip sample preconcentration before separation, a polarityswitching technique was employed to achieve subnanomolar detection limits for many peptide standards [38],... 

D polyacrylamide gel electrophoresis (2D PAGE) and MS are weU-established and the most commonly employed techniques in proteomics today. 2D PAGE, however, provides limited information of the total amount of proteins. Low-abimdance proteins and small peptides are not detected [1]. Additional methodologies and techniques in sample preparation, selective enrichment, high resolution separation, and detection need to be developed which would allow even higher resolution than 2D PAGE. Acceptable sensitivity to detect the low-abundance proteins is also still an issue. LC can address some of the above-mentioned... 

Capillary electrophoresis-MS (on-line coupling of CE to ESI-MS, CE-MS) provides high resolution separations of a wide range of proteins (Simo et al., 2004). Two-dimension electrophoresis (2-DE) and MS analysis allow resolution and identification of several thousand proteins. The procedure, which can be automated, involves excision of the protein spots from the 2-DE gel, followed by enzymatic digestion with a protease (e.g. trypsin) and MS analysis (Ashcroft, 2003). 2-DE polyacrylamide gel electrophoresis (2D-PAGE) provides resolution based on both the size and mass differences. 

---