Ubiquitylation is an important posttranslational protein modification that is. Despite the diverse biological pathways known to be regulated by ubiquitylation, global identification of substrates that are targeted for ubiquitylation has remained a challenge. Annotated msms spectra for all ubiquitinmodified peptides identified. Proteomewide identification of ubiquitin interactions. Characterization of the ubiquitinmodified proteome. The sequest search algorithm version 28 was used to search ms ms spectra against a concatenated targetdecoy database. The thermo tune software was set in such a way that in highest. Ubiquitin digly proteomics as an approach to identify and quantify. Sitespecific quantification of protein ubiquitination on ms2. The finding that k27linked ubiquitin was resistant to usp2cc, together with the potential protection of ubiquitin chains from cleavage by endogenous ubiquitinbinding proteins, suggested that this analysis may underrepresent the ubiquitylated portion of the diglymodified proteome. Gg sites were shown to be the result of ubiquitination as opposed to nedd8ylation or isg15ylation 59, 60. However, in a recent study using the same methodology as used in the current work, 94% of k. The data collected includes not only links to publications in pubmed, but also provides information about sample types, species, and experimental conditions.
Indepth analysis of the ubiquitinome using mass spectrometry will lead to a. Ubiquitin digly proteomics as an approach to identify. To globally characterize the human ubiquitinmodified proteome ubiquitinome, we utilized a monoclonal antibody that recognizes diglycine diglycontaining isopeptides following trypsin. Quantitative analysis of ubiquitinated proteins in human. Mass spectrometrybased proteomics for investigating. Proteomicsbased analysis of ubiquitin modification sites in proteins. Ubiquitylation is an important posttranslational protein modification that is involved. Ggmodified peptides to 272 proteins that fulfilled our selection criteria of fivefold increase and p isg15. Using the ubiquitinmodified proteome to monitor distinct. Using the ubiquitinmodified proteome to monitor distinct and spatially restricted protein homeostasis dysfunction. Proteomewide identification and functional analysis of. Protein ubiquitylation is one of the most prevalent posttranslational modifications ptm within cells. Systematic and quantitative assessment of the ubiquitinmodified proteome.
Especially, when multiple modifications occur on a single peptide. Ubiquitylation is a posttranslational protein modification that. Proteome software discoverybased msms, proteomics and. Modification of proteins with the 76 amino acid protein ubiquitin plays. Here, we demonstrate that the ubmodified proteome can exceed the. Proteomewide quantitative analysis of protein ubiquitination is important to gain. Ubiquitin modification of target lysine residues typically marks. The finding that k27linked ubiquitin was resistant to usp2cc, together with the potential protection of ubiquitin chains from usp2ccmediated cleavage by endogenous ubiquitinbinding proteins, suggested that this analysis may underrepresent the ubiquitylated portion of the diglymodified proteome.
Typically, ubiams allows the identification of dozens to hundreds of ubiquitin interactors from any type of cell lysate, and can be used to study cell type or stimulusdependent ubiquitin. Rational designs exploiting the chemistry of ubiquitin for enriching ubiquitinmodified proteins peptides. Protein ubiquitination is an important posttranslational modification. Ubiquitin modification of target lysine residues typically. Characterization of the ubiquitinmodified proteome regulated by.
Work in the zhang lab was supported by the hundred talents program c of the. Gendron jm1, webb k1, yang b1, rising l1, zuzow n1, bennett ej2. Using the ubiquitinmodified proteome to monitor distinct and. The proteins and ubiquitination sites were identified using maxquant software version 1. Proteomewide identification and functional analysis of ubiquitinated. To globally characterize the human ubiquitinmodified proteome ubiquitinome, we utilized a monoclonal antibody that recognizes diglycine diglycontaining isopeptides following trypsin digestion. Ub is a component of a multipathway intracellular proteolytic system called the ub system, or the ub proteasome system. R software gene ontology go analysis of 108 ubiquitinated proteins. Here, we demonstrate that the ubmodified proteome can exceed the sensitivity. However, consistent with isg15 expression occurring only in response to interferon ifn. Systematic and quantitative assessment of the ubiquitin. Most of the ubdependent pathways involve processive degradation of ubconjugated ubiquitylated proteins by the 26s proteasome, an atpdependent multisubunit protease.
Global analysis of the ubiquitinmodified proteome using mass spectrometry. Improvement of ubiquitylation site detection by orbitrap mass. Quantification was performed using an inhouse software itmsq,34. Like ubiquitin, two related ublsnedd8 and isg15contain cterminal digly motifs generated by trypsin cleavage, and the diglymodified proteome therefore represents a composite of proteins modified by these three ubls.