Internal amino acid sequence analysis of proteins separated by oneor two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose

http://www.pnas.org/content/84/20/6970.full.pdf
We have developed a general two-step method for obtaining peptide fragments for sequence analysis from
picomole quantities of proteins separated by gel electrophoresis. After separation by one- or two-dimensional polyacrylamide gel electrophoresis, proteins are electrophoretically transferred (electroblotted) onto nitrocellulose, the protein-containing regions are detected by reversible staining and are cut out,
and each protein is digested in situ by proteolytic enzymes such
as trypsin or staphylococcal V-8 protease. The resulting peptide
fragments are separated by narrow-bore reverse-phase HPLC,
collected, and sequenced in a gas-phase sequenator. Excellent
peptide recoveries and the absence of extraneous contaminants
in the separation of the peptide fragment mixture allow the
generation of extensive internal sequence information from
picomole amounts of protein. The method thus overcomes the
problem of obtaining amino acid sequence data from Nterminally blocked proteins and provides multiple, independent stretches of sequence that can be used to generate
oligonucleotide probes for molecular cloning and/or used to
search sequence data bases for related proteins. This method
has been successfully applied to the routine amino acid sequence analysis of a wide range of proteins isolated from oneand two-dimensional polyacrylamide gels.

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