Journal
JOURNAL OF PROTEOME RESEARCH
Volume 8, Issue 7, Pages 3702-3711Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr900257z
Keywords
Azide; azidohomoalanine; azide-alkyne cycloaddition; cross-linking; cyclooctyne; enrichment; mass spectrometry; proteomics; peptides; pulse-chase labeling
Categories
Funding
- ECHO-project
- Netherlands Organization for Scientific Research (MVO)
- National Institutes of Health [GM058867]
- National Science Foundation
- National Defense Science and Engineering
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A general method is described to sequester peptides containing azides from complex peptide mixtures, aimed at facilitating mass spectrometric analysis to study different aspects of proteome dynamics. The enrichment method is based on covalent capture of azide-containing peptides by the azide-reactive cyclooctyne (ARCO) resin and is demonstrated for two different applications. Enrichment of peptides derived from cytochrome c treated with the azide-containing cross-linker bis(succinimidyl)-3-azidomethyl glutarate (BAMG) shows several cross-link containing peptides. Sequestration of peptides derived from an Escherichia coli proteome, pulse labeled with the bio-orthogonal amino acid azidohomoalanine as substitute for methionine, allows identification of numerous newly synthesized proteins. Furthermore, the method is found to be very specific, as after enrichment over 87% of all peptides contain (modified) azidohomoalanine.
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