Microwave assisted acid cleavage for denaturation and proteolysis of intact human adenovirus

Abstract

Microwave assisted acid cleavage was applied directly to intact adenovirus type 5 to achieve denaturation and proteolysis in a single reaction. The speed of the digestion, coupled with the simplicity of MALDI analysis, allowed peptide products to be profiled in less than 5 min. Identification of peptides from a range of proteins by MALDI-TOF confirms that both denaturation and proteolysis were achieved using low concentrations of acetic acid (12.5%) and short incubations (1.5–2 min) at high temperatures (140 °C). These conditions favor production of peptides that carry Asp on their C-termini. When this cleavage reaction is carried out in highly enriched H 2 18O, a single atom of 18O is introduced site-specifically into the carboxyl terminal. The labeling reaction is applied to label reporter peptides from human adenovirus type 5 harvested from HeLa cells. Small peptide products of endogenous processing were also observed.