Proteomics

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Improvement of ubiquitination site detection by Orbitrap mass spectrometry.


ABSTRACT: We describe a modified workflow to enrich for and detect diGly peptides originating from ubiquitinated proteins using immunopurification. Using a combination of several relatively simple modifications in the sample preparation and mass spectrometric detection protocols, we now routinely detect over 24,000 diGly modified peptides in a single sample. We show the efficacy of this strategy for cell lysates from both non-labeled and metabolically labeled (SILAC) mammalian cells. Furthermore, we demonstrate that this optimized strategy is also useful for the in-depth identification of the endogenous, unstimulated ubiquitinome of in vivo samples such as mouse brain tissue. As such, this study presents an addition to the toolbox of the ubiquitination site analysis for the identification of the deep ubiquitinome.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Jeroen Demmers  

LAB HEAD: Jeroen Demmers

PROVIDER: PXD007101 | Pride | 2017-11-14

REPOSITORIES: Pride

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Publications

Improvement of ubiquitylation site detection by Orbitrap mass spectrometry.

van der Wal Lennart L   Bezstarosti Karel K   Sap Karen A KA   Dekkers Dick H W DHW   Rijkers Erikjan E   Mientjes Edwin E   Elgersma Ype Y   Demmers Jeroen A A JAA  

Journal of proteomics 20171106


Ubiquitylation is an important posttranslational protein modification that is involved in many cellular events. Immunopurification of peptides containing a K-ε-diglycine (diGly) remnant as a mark of ubiquitylation combined with mass spectrometric detection has resulted in an explosion of the number of identified ubiquitylation sites. Here, we present several significant improvements to this workflow, including fast, offline and crude high pH reverse-phase fractionation of tryptic peptides into o  ...[more]

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