Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases
Autor: | John T. Stults, Christopher Grimley, Susan C. Wong, Todd M. Billeci, William J. Henzel, Colin K. Watanabe |
---|---|
Rok vydání: | 1993 |
Předmět: |
Databases
Factual Molecular Sequence Data Peptide computer.software_genre Mass Spectrometry Protein sequencing Peptide mass fingerprinting Escherichia coli Humans Electrophoresis Gel Two-Dimensional Trypsin Amino Acid Sequence Cloning Molecular Peptide sequence chemistry.chemical_classification Gel electrophoresis Multidisciplinary Chromatography Sequence Homology Amino Acid Database Sequence database Edman degradation Proteins Peptide Fragments Recombinant Proteins Molecular Weight chemistry Biochemistry Growth Hormone Bottom-up proteomics computer Research Article |
Zdroj: | Proceedings of the National Academy of Sciences. 90:5011-5015 |
ISSN: | 1091-6490 0027-8424 |
DOI: | 10.1073/pnas.90.11.5011 |
Popis: | A rapid method for the identification of known proteins separated by two-dimensional gel electrophoresis is described in which molecular masses of peptide fragments are used to search a protein sequence database. The peptides are generated by in situ reduction, alkylation, and tryptic digestion of proteins electroblotted from two-dimensional gels. Masses are determined at the subpicomole level by matrix-assisted laser desorption/ionization mass spectrometry of the unfractionated digest. A computer program has been developed that searches the protein sequence database for multiple peptides of individual proteins that match the measured masses. To ensure that the most recent database updates are included, a theoretical digest of the entire database is generated each time the program is executed. This method facilitates simultaneous processing of a large number of two-dimensional gel spots. The method was applied to a two-dimensional gel of a crude Escherichia coli extract that was electroblotted onto poly(vinylidene difluoride) membrane. Ten randomly chosen spots were analyzed. With as few as three peptide masses, each protein was uniquely identified from over 91,000 protein sequences. All identifications were verified by concurrent N-terminal sequencing of identical spots from a second blot. One of the spots contained an N-terminally blocked protein that required enzymatic cleavage, peptide separation, and Edman degradation for confirmation of its identity. |
Databáze: | OpenAIRE |
Externí odkaz: |
načítá se...