Dataset Information

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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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]

PMID: 29122726

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Project description:Huntington’s disease is caused by a polyglutamine repeat expansion at the N-terminus of the huntingtin protein which affects the function and folding of the protein, and results in intracellular protein aggregates. Here, we examined whether this mutation leads to altered ubiquitination of huntingtin and other proteins in both soluble and insoluble fractions of brain lysates of the Q175 knock-in Huntington disease mouse model and the Q20 wild-type mouse model. Ubiquitination sites are detected by identification of Gly-Gly (diGly) remnant motifs that remain on modified lysine residues after digestion. We identified K6, K9, K132, K804 and K837 as endogenous ubiquitination sites of soluble huntingtin, with wild-type huntingtin being mainly ubiquitinated at K132, K804 and K837. Mutant huntingtin protein levels were strongly reduced in the soluble fraction while K6 and K9 were mainly ubiquitinated. In the insoluble fraction increased levels of huntingtin K6 and K9 diGly sites were observed for mutant huntingtin as compared to wild type. Besides huntingtin, proteins with various roles, including membrane organization, transport, mRNA processing, gene transcription, translation, catabolic processes and oxidative phosphorylation, were differently expressed or ubiquitinated in wild-type and mutant huntingtin brain tissues. Correlating protein and diGly site fold-changes in the soluble fraction revealed that diGly site abundances of the majority of the proteins were not related to protein fold-changes, indicating that these proteins were differentially ubiquitinated in the Q175 mice. In contrast, both the fold-change of the protein level and diGly site level were increased for several proteins in the insoluble fraction, including ubiquitin, ubiquilin-2, sequestosome-1/p62 and myo5a. Our data sheds light on putative novel proteins involved in different cellular processes as well as their ubiquitination status in Huntington’s disease, which forms the basis for further mechanistic studies to understand the role of differential ubiquitination of huntingtin as well as ubiquitin-regulated processes in Huntington’s disease.

Dataset Information

Improvement of ubiquitination site detection by Orbitrap mass spectrometry.

Publications

Improvement of ubiquitylation site detection by Orbitrap mass spectrometry.

Similar Datasets

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