Sutton, C. W., Pemberton, K. S., Cottrell, J. S., Corbett, J. M., Wheeler, C. H., Dunn, M. J., Pappin, D. J. (1995) Identification of myocardial proteins from two-dimensional gels by peptide mass fingerprinting. Electrophoresis, 16 (3). pp. 308-316. ISSN 01730835 (ISSN)
Abstract
Two-dimensional gels offer a powerful method for separating complex protein mixtures, but subsequent methods for analysing individual components, such as protein sequencing and Western immunoblotting, are laborious and slow. The identification of proteins can be accelerated by using a combination of protease digestion and matrix assisted laser desorption-mass spectrometry (MALDI-MS). The peptide mass spectrum of a protein represents a unique fingerprint determined by the amino acid sequence and the cleavage properties of the protease. Software has been developed so that peptide masses can be used to search a mass-based peptide database generated from established protein sequence databases. A list of the closest matching proteins is produced to allow identification of the sample. The strategy was applied to 52 protein spots from human myocardial tissue separated by two-dimensional electrophoresis (2-DE) gels and analysed blind. Conditions for optimal trypsin digestion of proteins electroblotted onto polyvinylidene difluoride (PVDF) membranes are described. Mass data were generated from both Coomassie Brilliant Blue and sulforhodamine B-stained proteins, though the former required destaining prior to digestion. Alkylation of cysteine and oxidation of methionine were significant modifications that influenced the successful identification of a protein spot. Examples are presented to illustrate the advantages and disadvantages of this approach.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | databases matrix assisted laser desorption-mass spectrometry myocardial proteins peptide fingerprinting two-dimensional polyacrylamide gel electrohoresis conference paper gel electrophoresis heart human human tissue mass spectrometry methodology peptide mapping protein analysis Electrophoresis, Gel, Two-Dimensional Myocardium Proteins Spectrum Analysis, Mass Support, Non-U.S. Gov't Trypsin |
| Subjects: | bioinformatics > genomics and proteomics > design > protein network design > peptide design bioinformatics > genomics and proteomics > design > protein design organism description > animal > mammal > primates > hominids > human |
| CSHL Authors: | |
| Communities: | CSHL labs > Pappin lab |
| Depositing User: | Jessica Koos |
| Date: | 1995 |
| Date Deposited: | 12 Aug 2014 16:04 |
| Last Modified: | 12 Aug 2014 16:04 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/30644 |
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