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R. M. KAMP and H. HIRANO
4
Sequential Deblocking of Proteins
The above mentioned deblocking enzymatic and chemical methods may be used
in combination to allow step-by-step deblocking for sequencing of proteins with
unknown blocking groups, which are immobilized on a PVDF membrane
(Hirano 1997). The protein sample after separation on polyacrylamide gel is
transferred onto a PVDF membrane by electroblotting and subjected to gasphase sequencing.
If sequencing does not show results, the membrane is removed from the
sequencer and treated with TFA vapor to remove the acetyl groups of acetylserine
and acetylthreonine. After this deblocking reaction, the PVDF membrane is
applied to the sequencer. If sequencing fails, the membrane is incubated in
diluted Hel to remove formyl groups and then subjected to protein sequencer. If
the protein is N-blocked with formyl-group, the proteins can be sequenced.
When not, the sample is applied for pyroglutamyl peptidase digestion to remove
N-terminal pyroglutamic acid. If sequencing after above treatment was impossiElectrop,horesis
I I.
B ottmg
I
Sequencing
First Deblocking
Sequencing
Second Deblocking
Sequencing
Third Deblocking
Sequencing
Fourth Deblocking
Sequencing
Deblocking
Fig. 22.7. Step-by-step deblocking for N-terminally blocked proteins electroblotted onto a PVDP
membrane by combined chemical and enzymatic methods