Electrophoresis-based methods, PAGE

Fig. 1. Difference gel electrophoresis (DICE) workflow. An aliquot of each sample is mixed to create the internal standard. All samples and the standard are labeled, and then combined before running the 2-D gel. Image acquisition and analysis complete the workflow. Obtained with permission from Westermeier R, Scheibe B. Difference gel electrophoresis based on lys/cys tagging. In Posch A. Ed. Sample Preparation for 2D PAGE. Methods in Molecular Biology, Humana Press, Totowa, NJ (2007).

The second step in 2D electrophoresis is to separate proteins based on molecular weight using SDS-PAGE. Individual proteins are then visualized by Coomassie or silver staining techniques or by autoradiography. Because 2D gel electrophoresis separate proteins based on independent physical characteristics, it is a powerful means to resolve complex mixtures proteins (Fig. 2.1). Modem large-gel formats are reproducible and are the most common method for protein separation in proteomic studies. 

Size-based analysis of SDS-protein complexes in polyacrylamide gels (SDS-PAGE) is the most common type of slab gel electrophoresis for the characterization of polypeptides, and SDS-PAGE is one of the most commonly used methods for the determination of protein molecular masses.117 The uses for size-based techniques include purity determination, molecular size estimation, and identification of posttranslational modifications.118119 Some native protein studies also benefit from size-based separation, e.g., detection of physically interacting oligomers. 

For many years, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) methods have been used as an essential tool to determine the hydrodynamic size, monitor product purity, detect minor product or process-related impurities, and confirm batch-to-batch consistency of protein and antibody products. ITowever, gel-based techniques have several limitations, such as lack of automation, varying reproducibility, and a limited linear range. SDS-PAGE is also labor-intensive and generates large volume of toxic waste. Most importantly, the technique does not provide quantitative results for purity and impurity determination of proteins and antibodies. 

Regardless of the chromatography method that is used, fractions containing subsets of the proteins in the sample are collected at the bottom of the column. Each of the fractions is assayed for enzyme activity. In addition, the complexity of the fraction is evaluated by separating the proteins in a small sample of the fraction using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This method separates proteins in mixtures based on their size. 

The high sample demands and low-throughput of LC-MS methods have led to the creation of a capillary electrophoresis (CE) platform for ABPP [48]. Proteomes are labeled with a fluorescent probe, digested with trypsin, and enriched with antifluorophore antibody resins. Use of CE coupled with laser-induced fluorescence (LIF) detection to analyze the enriched peptides resulted in far superior resolution to ID SDS-PAGE, particularly for enzymes that share similar molecular masses. Sensitivity limits of 0.05-0.1 pmol/mg proteome, negligible sample requirements (—0.01—0.1 pg proteome), and the ability to perform rapid CE runs in parallel with 96-channel instruments, make CE-based ABPP a potentially powerful technique. One drawback is that the identities of the probe-labeled proteins are not immediately apparent, and correlated LC-MS experiments must be performed to assign protein identities to the peaks on the CE readout. 

---