Microsequence analysis of the N-terminally blocked proteins immobilized on polyvinylidene difluoride membrane by western blotting.
A technique has been developed for efficient deblocking and subsequent microsequencing of N-terminally blocked proteins immobilized on a polyvinylidene difluoride (PVDF) membrane at the picomole levels. In this technique, proteins were first separated by polyacrylamide gel electrophoresis and then transferred onto a PVDF membrane by Western blotting. The electroblotted proteins with N-terminal acetylserine or acetylthreonine could be deblocked on-membrane by treatment with trifluoroacetic acid vapor and sequenced by a gas-phase protein sequencer. Similarly, N-formylated proteins could be deblocked on-membrane in HC1 solution and then directly sequenced from the N-terminal amino acid. Proteins with N-terminal pyroglutamic acid were enzymatically deblocked by in situ pyroglutamyl peptidase digestion, and N-acetylated proteins were also enzymatically deblocked with acylamino acid-releasing enzyme (AARE) after on-membrane digestion with trypsin to generate the N-terminal peptide fragment. This tryptic digestion was required since AARE can remove the acetylamino acid only from a short peptide. Based on these four deblocking methods, we present a strategy for sequential deblocking and subsequent N-terminal sequence analysis of N-blocked protein immobilized on PVDF membrane.
Hirano
H
H
National Institute of Agrobiological Resources, Ibaraki, Japan.
Komatsu
S
S
Kajiwara
H
H
Takagi
Y
Y
Tsunasawa
S
S
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003574:Cytochrome c Group / Q000737:chemistry; D005848:Geotrichum / Q000201:enzymology; D008049:Lipase / Q000737:chemistry; D008567:Membranes, Artificial; D008832:Microchemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011145:Polyvinyls; D011506:Proteins / Q000737:chemistry*; D011761:Pyrrolidonecarboxylic Acid; D017421:Sequence Analysis; D012694:Serine; D013482:Superoxide Dismutase / Q000737:chemistry; D013912:Threonine; D014357:Trypsin / Q000378:metabolism; D014367:Tryptophan Synthase / Q000737:chemistry; D005465:Fluorocarbon Polymers
D003574:Cytochrome c Group; D008049:Lipase; D008567:Membranes, Artificial; D010446:Peptide Fragments; D011145:Polyvinyls; D011506:Proteins; D011761:Pyrrolidonecarboxylic Acid; D012694:Serine; D013482:Superoxide Dismutase; D013912:Threonine; D014357:Trypsin; D014367:Tryptophan Synthase; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers