Systematic peptide fragmentation of polyvinylidene difluoride(PVDF)-immobilized proteins prior to microsequencing

The sequential in situ digestion of proteins immobilized on polyvinylidene difluoride (PVDF) has been systematically designed and optimized. The method consists of immobilization of the proteins on PVDF, S-carboxymethylation, and then successive in situ digestions using specific proteases. In order to obtain high yields of the peptide fragments, from which specific amino acid residues connected to the N- or C-terminal of the resulting digestion fragments can be deduced, the cleavages are performed in the following order: (1) Achromobacter protease I (API), (2) endoproteinase Asp-N, and (3) trypsin. Procedures for recovering the numerous fragments remaining on the PVDF membrane after the third digestion with trypsin are also discussed. Application of sequential in situ digestion for the acquisition of fragments suitable for sequencing from digests of large-molecular-weight proteins is also presented.