MICROSEQUENCE ANALYSIS OF Nα-BLOCKED PROTEINS ELECTROBLOTTED ONTO AN IMMOBILIZING MATRIX FROM POLYACRYLAMIDE GELS

Abstract

The method for direct microsequence analysis of N^α-blocked proteins electroblotted onto polyvinylidene difluoride (PVDF) membranes from polyacrylamide gels was developed. The method consists of the following steps. N^α-Acetylated and pyroglutamated proteins (>32 picomoles) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto the PVDF membranes. The portion of the membrane carrying the protein band was cut out and treated with polyvinylpyrroiidone (PVP)-40 in acetic acid solution to block the unbound protein-binding sites on the membrane. Subsequently, the proteins with N-terminal pyroglutamic acid were directly digested on the membrane with pyroglutamyl peptidase to remove the blocked residue, and sequenced by a gas-phase sequencer. On the other hand, the N^α-acetylated proteins were digested on the membrane with trypsin, the resultant tryptic peptides were released out of the membrane, the N-terminal peptide was efficiently deblocked with acylamino acid-releasing enzyme and sequenced by the gas-phase sequencer.