Leupeptins; Peptides; Proteome; Ubiquitin; Lysine; benzyloxycarbonylleucyl-leucyl-leucine aldehyde; Alkylation; Amino Acid Sequence; Animals; Cell Line, Tumor; Chromatography, Liquid; Humans; Leupeptins/pharmacology; Lysine/metabolism; Mass Spectrometry/methods; Mice; Peptides/analysis; Reference Standards; Software; Ubiquitin/analysis; Ubiquitin/chemistry; Mass Spectrometry; Neuroscience (all); Chemical Engineering (all); Biochemistry, Genetics and Molecular Biology (all); Immunology and Microbiology (all); General Immunology and Microbiology; General Biochemistry, Genetics and Molecular Biology; General Chemical Engineering; General Neuroscience
Résumé :
[en] Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The protocol outlined here takes advantage of the di-glycine (-GG) modification left after the tryptic digestion of ubiquitin incorporated in a chain. By quantifying these topology-characteristic peptides the relative abundance of each ubiquitin chain topology can be determined. The steps required to quantify these peptides by a parallel reaction monitoring experiment are reported taking into consideration the stabilization of ubiquitin chains. Preparation of heavy controls, cell lysis, and digestion are described along with the appropriate mass spectrometer setup and data analysis workflow. An example data set with perturbations in ubiquitin topology is presented, accompanied by examples of how optimization of the protocol can affect results. By following the steps outlined, a user will be able to perform a global assessment of the ubiquitin topology landscape within their biological context.
Disciplines :
Biochimie, biophysique & biologie moléculaire
Auteur, co-auteur :
LONGWORTH, Joseph ; Quantitative Biology Unit, Luxembourg Institute of Health
Mendes, Marta L; Quantitative Biology Unit, Luxembourg Institute of Health
DITTMAR, Gunnar ; Quantitative Biology Unit, Luxembourg Institute of Health, Gunnar.Dittmar@lih.lu
Co-auteurs externes :
yes
Langue du document :
Anglais
Titre :
Ubiquitin Chain Analysis by Parallel Reaction Monitoring.
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