Internal Amino Acid Sequence Analysis of Proteins Separated by One-or Two-Dimensional Gel Electrophoresis after in situ Protease Digestion on Nitrocellulose

• Published in: Proceedings of the National Academy of Sciences - PNAS 1987-10, Vol.84 (20), p.6970-6974
• Main Authors: Aebersold, Ruedi H., Leavitt, John, Saavedra, Raul A., Hood, Leroy E., Stephen B. H. Kent
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Cellulose nitrate; Chromatography, High Pressure Liquid; Collodion; Electrophoresis; Electrophoresis, Polyacrylamide Gel - methods; Enzymes; Fundamental and applied biological sciences. Psychology; gel electrophoresis; Gels; General aspects, investigation methods; Laboratory staining techniques; Membranes, Artificial; nitrocellulose; Peptide Fragments - isolation & purification; Peptide Hydrolases; Peptide Mapping; Protein digestion; Protein sequence analysis; Proteins; Proteins - analysis; Sequence analysis
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.84.20.6970

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 N-terminally 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 one- and two-dimensional polyacrylamide gels.