Deblocking and Subsequent Microsequence Analysis of Nα-Blocked Proteins Electroblotted onto PVDF Membrane1

Abstract

A method was developed for direct microsequencing of N^α-acetylated proteins electroblotted onto polyvinylidene difluoride membranes from polyacrylamide gels. N^α-Acetylated proteIns (>32 pmol), 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% polyvlnylpyrrolidone in acetic acid solution at 37°C for 30 mm. The protein was digested on the membrane with 5–10 μg of trypsin at 3TC for 24 h. During tryptic digestion, the resultant peptides were released from the membrane and the N-terminal peptide was efficiently deblocked with 50 mU of acylamino acid-releasing enzyme at 37°C for 12 h. Picomole levels of the deblocked proteins could be sequenced directly by use of a gas-phase protein sequencer.