Microsequence analysis of the N‐terminally blocked proteins immobilized on polyvinylidene difluoride membrane by Western blotting

Abstract

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 electro‐blotted 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.