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...

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Bibliographic Details
Published in:Journal of biochemistry (Tokyo) 1996-07, Vol.120 (1), p.29-34
Main Authors: Iwamatsu, A, Yoshida-Kubomura, N
Format: Article
Language:English
Subjects:
Amino Acid Sequence
amino acid sequences
amino acid sequencing
Carrier Proteins - chemistry
Chromatography, High Pressure Liquid
deducible amino acid residues
Endopeptidases
Granulocyte Colony-Stimulating Factor - chemistry
immobilization
Membranes, Artificial
Metalloendopeptidases
Methylation
Molecular Sequence Data
Peptide Fragments - chemistry
Peptide Mapping
Polyvinyls
proteinases
proteins
proteolysis
PVDF membrane
Recombinant Proteins
recoveries of digests from PVDF
S-alkylation of protein
Sequence Analysis - methods
sequential in situ digestion
Serine Endopeptidases
Trypsin
Zona Pellucida
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Summary: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.
ISSN:0021-924X
1756-2651
DOI:10.1093/oxfordjournals.jbchem.a021389