DEBLOCKING AND SUBSEQUENT MICROSEQUENCE ANALYSIS OF N-ALPHA-BLOCKED PROTEINS ELECTROBLOTTED ONTO PVDF MEMBRANE

A method was developed for direct microsequencing of N(alpha)acetylated proteins electroblotted onto polyvinylidene difluoride membranes from polyacrylamide gels. N(alpha)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% polyvinylpyrrolidone in acetic acid solution at 37-degrees-C for 30 min. The protein was digested on the membrane with 5-10-mu-g of trypsin at 37-degrees-C 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-degrees-C for 12 h. Picomole levels of the deblocked proteins could be sequenced directly by use of a gas-phase protein sequencer.