UBC Theses and Dissertations
Analysis of proteins isolated from dissolvable polyacrylamide gels using mass spectrometry Ramanowski, Peter
Polyacrylamide gel electrophoresis is a simple yet powerful means of resolving complex mixtures of proteins and is used across many fields of research, including proteomics. The in-gel digestion has become the staple tool for identification of proteins run on SDS-PAGE gels but has unfortunate shortcomings such as unpredictable sequence coverage and the inability to obtain precise intact protein masses. Other methods such as electro-elution and passive diffusion evade these limitations by recovering intact protein but have difficulties isolating protein of high molecular weight. In this study, the use of a dissolvable SDS-PAGE gel for the isolation of intact proteins and subsequent analysis by mass spectrometry was evaluated. The resolutions of standard bis-acrylamide (BIS) crosslinked gels were compared to dissolvable gels crosslinked with bis-acrylylcystarnine (BAC) to show virtually identical resolving capabilities. The model protein a-casein was successfully extracted from BACcrosslinked gels along with several other standard proteins. Accurate intact mass measurements and specific enzymatic processing were done to show a mass shift corresponding to 8 phosphorylation sites. In addition, gel-extracted protein was digested in solution with several enzymes, including trypsin, to demonstrate higher peptide recoveries and sequence coverage compared to the widely used in-gel digestion approach. In light of these additional features which are not available with standard gels, such as the ability to obtain precise protein masses and increased sequence coverage when digested, BAC-crosslinked gels may well become a reasonable alternative for proteomics analyses.
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