Selective enrichment of azide-containing peptides from complex mixtures.
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.
Nessen
Merel A
MA
Mass Spectrometry of Biomacromolecules, Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018WV Amsterdam, The Netherlands.
Kramer
Gertjan
G
Back
JaapWillem
J
Baskin
Jeremy M
JM
Smeenk
Linde E J
LE
de Koning
Leo J
LJ
van Maarseveen
Jan H
JH
de Jong
Luitzen
L
Bertozzi
Carolyn R
CR
Hiemstra
Henk
H
de Koster
Chris G
CG
D000409:Alanine / Q000031:analogs & derivatives / Q000737:chemistry; D000595:Amino Acid Sequence; D001386:Azides / Q000737:chemistry*; D002412:Cations; D002852:Chromatography, Ion Exchange / Q000379:methods; D003432:Cross-Linking Reagents / Q000494:pharmacology; D045304:Cytochromes c / Q000737:chemistry; D005977:Glutarates / Q000737:chemistry; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D020543:Proteome; D040901:Proteomics / Q000379:methods
D001386:Azides; D002412:Cations; D003432:Cross-Linking Reagents; D005977:Glutarates; D010455:Peptides; D020543:Proteome; C506098:azidohomoalanine; D045304:Cytochromes c; D008715:Methionine; D000409:Alanine