(342e) Acid-Degradable Polyacrylamide Gel Electrophoresis for Isolation of Structurally and Functionally Intact Proteins and Its Implications

In post-genomic era, understanding protein structure/function coupled with genomics, called proteomics, is an emerging technology not only in understanding biology at a molecular level but also in discovering biomarkers for diagnosis and therapy. Pivotal steps in current proteomic studies are to separate proteins from a highly complex biological sample and to analyze the isolated proteins using various analysis techniques such as MALDI (matrix-assisted laser desorption/ionization). Polyacrylamide gel electrophoresis (PAGE), conventionally applied with or without sodium dodecyl sulfate (SDS) in denatured or native PAGE, respectively, has been the predominant technique in simultaneously separating a number of proteins. However, significantly larger size of proteins than the pores in a polyacrylamide gel makes recovering the separated proteins in their structurally and functionally native forms extremely inefficient or almost impossible, particularly for large proteins. Frequently, the proteins are fragmented by proteolysis in order to be released by diffusion (in-gel digestion), resulting in obstructed elucidation of native structure-function relationships of a separated protein. In this study, we tested the novel idea of obtaining structurally and functionally intact proteins by degrading polyacrylamide gel (rather than fragmenting separated proteins) under a mildly acidic condition. A model large protein, mouse IgG1 antibody (MW 150 kDa) was electrophoresed in the polyacrylamide gel cross-linked by acid-cleavable ketal diacrylamide instead of conventionally used methylene bisacrylamide, under native PAGE conditions. A protein band identified at 150 kDa was excised and hydrolyzed at the acidic solution consisting of formic acid and propanol in water. The proteins were further purified by selectively precipitating hydrolyzed polyacrylamide. The final amount of the isolated proteins quantified by enzyme-linked immunosorbent assay (ELISA) demonstrated that approximately 70% of the antibodies were recovered from the degradable polyacrylamide gel, while only a traceable amount of the antibodies were obtained from a conventionally used nondegradable polyacrylamide gel. The preserved specific binding capability of the isolated antibodies to target antigens as well as intact molecular weights proved the successful isolation of structurally and functionally native proteins. This novel technology can be used to isolate other types of macromolecules, macromolecular complexes, and nanomaterials.