Relative protein quantification by isobaric SILAC with immonium ion splitting (ISIS)

Détails

ID Serval
serval:BIB_173947FB7885
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Institution
Titre
Relative protein quantification by isobaric SILAC with immonium ion splitting (ISIS)
Périodique
Molecular and Cellular Proteomics
Auteur(s)
Colzani M., Schutz F., Potts A., Waridel P., Quadroni M.
ISSN
1535-9484 (Electronic)
Statut éditorial
Publié
Date de publication
2008
Peer-reviewed
Oui
Volume
7
Numéro
5
Pages
927-937
Langue
anglais
Résumé
Metabolic labeling techniques have recently become popular tools for the quantitative profiling of proteomes. Classical stable isotope labeling with amino acids in cell cultures (SILAC) uses pairs of heavy/light isotopic forms of amino acids to introduce predictable mass differences in protein samples to be compared. After proteolysis, pairs of cognate precursor peptides can be correlated, and their intensities can be used for mass spectrometry-based relative protein quantification. We present an alternative SILAC approach by which two cell cultures are grown in media containing isobaric forms of amino acids, labeled either with 13C on the carbonyl (C-1) carbon or 15N on backbone nitrogen. Labeled peptides from both samples have the same nominal mass and nearly identical MS/MS spectra but generate upon fragmentation distinct immonium ions separated by 1 amu. When labeled protein samples are mixed, the intensities of these immonium ions can be used for the relative quantification of the parent proteins. We validated the labeling of cellular proteins with valine, isoleucine, and leucine with coverage of 97% of all tryptic peptides. We improved the sensitivity for the detection of the quantification ions on a pulsing instrument by using a specific fast scan event. The analysis of a protein mixture with a known heavy/light ratio showed reliable quantification. Finally the application of the technique to the analysis of two melanoma cell lines yielded quantitative data consistent with those obtained by a classical two-dimensional DIGE analysis of the same samples. Our method combines the features of the SILAC technique with the advantages of isobaric labeling schemes like iTRAQ. We discuss advantages and disadvantages of isobaric SILAC with immonium ion splitting as well as possible ways to improve it
Mots-clé
Amino Acids , analysis , Carbon Isotopes , Cell Line , Cell Line,Tumor , chemistry , Culture , Culture Media , Electrophoresis,Gel,Two-Dimensional , Genomics , Humans , Isoleucine , Isotope Labeling , Melanoma , metabolism , methods , Nitrogen Isotopes , Peptides , Proteins , Proteome , Proteomics , Switzerland , Valine
Pubmed
Web of science
Création de la notice
29/01/2009 23:14
Dernière modification de la notice
20/08/2019 13:47
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