Mass spectrometry analysis of K63-ubiquitinated targets in response to oxidative stress

The data described here provide the first large-scale analysis of lysine 63 (K63)-linked polyubiquitin targets. Protein ubiquitination is a prominent post-translational modification, and a variety of ubiquitin chains exists, serving a multitude of functions [1]. The chains differ by the lysine residue by which the ubiquitin monomers are linked. We used yeast Saccharomyces cerevisiae subjected to oxidative stress as a model to study K63 ubiquitination. K63 ubiquitinated targets were pulled-down by the K63-TUBE system (Tandem Ubiquitin Binding Entities) and analyzed by SILAC-based mass spectrometry [2]. The data are associated to the research article ‘K63 polyubiquitination is a new modulator of the oxidative stress response’ [3]. The mass spectrometry and the analysis dataset have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000960.


a b s t r a c t
The data described here provide the first large-scale analysis of lysine 63 (K63)-linked polyubiquitin targets. Protein ubiquitination is a prominent post-translational modification, and a variety of ubiquitin chains exists, serving a multitude of functions [1]. The chains differ by the lysine residue by which the ubiquitin monomers are linked. We used yeast Saccharomyces cerevisiae subjected to oxidative stress as a model to study K63 ubiquitination. K63 ubiquitinated targets were pulled-down by the K63-TUBE system (Tandem Ubiquitin Binding Entities) and analyzed by SILACbased mass spectrometry [2]. The data are associated to the research article 'K63 polyubiquitination is a new modulator of the oxidative stress response' [3]. The  Quantitative analysis using SILAC-based mass spectrometry under a physiologically important condition (oxidative stress).
1. Data, experimental design, materials and methods

Experimental design
Yeast Saccharomyces cerevisiae wild-type (WT) and the ubiquitin K63R mutant (K63R), which is unable to build K63 polyubiquitin chains, were subjected to H 2 O 2 treatment to induce oxidative stress. Proteins were extracted and K63-ubiquitinated targets were pulled-down prior to analysis by highresolution mass spectrometry. A SILAC-based mass spectrometry approach was used for quantification and differentiation between specific and unspecific interactions (Fig. 1).

Mass spectrometry analysis
Peptides were separated on a 15 cm Agilent ZORBAX 300 StableBond C18 column (75 μm ID, 3.5 μm particle, 300 Å pore size) by reverse-phase chromatography with a gradient of 5 to 40% acetonitrile over 160 min on an Eksigent NanoLC 2DPlus liquid chromatography system. LC-eluted peptides were injected inline onto an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific). Data-dependent analysis was performed using the top 20 most intense ions from each MS full scan. Dynamic exclusion was set to 90 seconds if mass to charge ratio (m/z) acquisition was repeated within a 45 seconds interval. MS1 data was collected in the FTMS mass analyzer at 60,000 resolution, in profile mode, and automatic gain control at 1E6. MS2 data was collected in the Ion Trap mass analyzer, in centroid mode, automatic gain control at 3E4, injection time of 100 ms, isolation window of 1 m/z, and normalized collision energy at 35%. Samples were injected three times to obtain technical replicates. The immunoprecipitation experiment was conducted twice to obtain two biological replicates (IPK63_1 and IPK63_2). To determine significance, data were compared to that from untreated cells (IPK63_Untreated). The deposited data (PXD000960) also contain two biological replicates (WCE1 and WCE2) from a SILAC-based mass spectrometry analysis of the Whole Cell Extract (cell lysate) prior to the K63 ubiquitin pulldown. The analyses compared the protein expression levels of the WT to the K63R strain, and each biological replicate was also injected three times as technical replicates to improve quantitation and coverage.

Data processing
The RAW data files were combined and processed using MaxQuant (version 1.3.0.5) matching against the yeast S. cerevisiae database (Uniprot, 2012 release). SILAC analyses were performed selecting Arg6 and Lys8 as heavy labels. Two missed cleavages were allowed, choosing trypsin as the proteolytic enzyme. Cysteine carbamidomethylation was selected as fixed, and diglycyl lysine, methionine oxidation, and Nterminal acetylation were selected as variable post-translational modification. Mass tolerance was set to 20 ppm for the FT mass analyzer and 0.5 Da for the Ion Trap mass analyzer. The false discovery rate (FDR) for protein, peptide, and site modifications was set to 1% based on the reverse yeast FASTA file. Minimum peptide length was seven, and every protein group was required to have at least one unique or razor peptide. Minimum ratio count for SILAC pairs was set to two. A complete set of parameters is provided in the MaxQuant results file deposited at ProteomeXchange. The results files (.zip) include two sets of MaxQuant tab delimited output tables (.txt) for the K63 ubiquitin immunoprecipitation (IPK63) and for the whole cell extract (WCE). The 'protein_groups', 'peptides', 'evidence', 'parameters' and 'summary' output files are presented here as Supplementary Tables 1-5, respectively, in Microsoft Excel spreadsheet format. Post-processing analyses eliminated contaminants and reversed sequences, and used only proteins present in both biological replicates, having at least two different peptides identified. Significance cut-off (1.57 fold change) was established based on the distribution of ratios (Light/Heavy) of the treated versus the untreated cells at 5% FDR.