Deblocking and Subsequent Microsequence Analysis of N^α-Blocked Proteins Electroblotted onto PVDF Membrane

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A method was developed for direct microsequencing of N^α-acetylated proteins electroblotted onto polyvinylidene difluoride membranes from polyacrylamide gels. N^α-Acetylated proteins (>32pmol), including horse heart cytochrome c, five mutants of yeast cytochrome c, and bovine erythrocyte superoxide dismutase, were separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. The portions of the membrane carrying the bands were cut out and treated with 0.5% polyvinylpyrrolidone in acetic acid solution at 37°C for 30min. The protein was digested on the membrane with 5-10μg of trypsin at 37°C for 24h. During tryptic digestion, the resultant peptides were released from the membrane and the N-terminal peptide was efficiently deblocked with 50mU of acylamino acid-releasing enzyme at 37°C for 12h. Picomole levels of the deblocked proteins could be sequenced directly by use of a gas-phase protein sequencer.