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 (greater than 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 micrograms 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.
Hirano
H
H
Institute for Protein Research, Osaka University.
Komatsu
S
S
Takakura
H
H
Sakiyama
F
F
Tsunasawa
S
S
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003574:Cytochrome c Group / Q000737:chemistry / Q000235:genetics; D004586:Electrophoresis; D006736:Horses; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D011145:Polyvinyls; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013482:Superoxide Dismutase / Q000737:chemistry; D005465:Fluorocarbon Polymers
D003574:Cytochrome c Group; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins; D013482:Superoxide Dismutase; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers