High-resolution separation, protein sample

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). 

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... 

Mass spectrometry has become a major player in proteome analysis because of its integration with high-resolution separation techniques and protein databases and its inherent high sensitivity, high structure specificity, high-mass capability, and opportunity for automation. Short analysis times and straightforward sample preparation steps are the other advantages of mass spectrometry-based proteomics. 

Although the viscosity of the sample solution may affect the resolution, for practical reasons highly concentrated protein samples will give the best separations in the case of SEC with respect to the process economy. Although the actual loading capacity depends on the separation problem and on the... 

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