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[…] The peptide mixture was analyzed in positive mode using a nanoAquity UPLC coupled LTQ Orbitrap Elite mass spectrometer (Thermo ). Chromatographic separation used a 2 cm trapping column (Acclaim PepMap 100) and a 15 cm EASY-spray analytical column (75 μm ID, C18 beads of 3.0 μm particle size, 100 Å pore size). The HPLC flow rate was set to 350 nL/min over a gradient of 1% buffer B (0.1% formic acid in acetonitrile) to 25% buffer B in 150 min. The full mass scan (300 to 2000 m/z) was acquired at a resolution of 120,000 with a maximum injection time of 500 ms, and MS/MS was performed in a data-dependent manner for the top 15 intense ions in the linear ion trap by collision-induced dissociation. Raw data were converted to mzXML format using ProteoWizard () and searched using the Crux pipeline () (version 2.1.16867) against the human UniProtKB/Swiss-Prot sequence database (downloaded on 2/20/15) (). Search parameters were set as the following: peptides between 6 and 25 amino acids long with a precursor mass tolerance of 0.5 amu, no missed cleavages, fully-enzymatic Arg-C digestion, a static propionyl modification (+56.026215) on lysines, and a maximum of 4 variable modifications consisting of up to 2 lysine ubiquitinations (+58.016716), 2 methylations (+14.01565), 2 dimethylations (−27.994915), 2 trimethylations (−13.979264), 2 acetylations (−14.015644), 1 methionine oxidation (+15.99492), and 1 STY phosphorylation (+79.966331). The mass of propionyl was subtracted from variable lysine modification masses (except methylation) due to the already applied static propionyl modification. For unpropionylated samples, the differing parameters were: up to 3 missed cleavages, fully-enzymatic trypsin digestion, no static modifications, and a maximum of 4 variable modifications consisting of up to 2 lysine ubiquitinations (+114.042931), 2 methylations (+14.01565), 2 dimethylations (+28.0313), 2 trimethylations (+42.046951), 2 acetylations (+42.010571), 1 methionine oxidation (+15.99492), and 1 STY phosphorylation (+79.966331). Prior to execution of the Percolator algorithm supplied by Crux, deltaCn scores were re-computed using an alternate definition: deltaCni = 1 – ((xcorr1-xcorri) / xcorr1). This adjustment was performed because the similar mass of trimethylation and acetylation results in identical xcorr values for the low mass accuracy MS/MS spectra from linear ion traps, which then led to invalid deltaCn values with the default equation used by Percolator. After application of a 5% FDR threshold, peptides were further filtered by ensuring they had the expected retention time relative to peptides having the identical unmodified sequence. We used the following procedure. First, peptides with the same unmodified sequence were sorted in ascending order by their Percolator PEP (posterior error probability). Then, each peptide (starting from lowest to highest PEP) was accepted if at least one of its MS/MS scan’s retention time was consistent relative to all currently accepted peptides having the same unmodified sequence. The expected relative retention time constraints were: ubiquitin < dimethyl ≤ trimethyl < acetyl < propionyl < methyl, oxidation < unmodified, and phosphorylation ≤ unmodified. Peptides expected to have the same retention times were allowed to elute within 2 min of each other. Finally, peptide H3K9me3 + K14ub was accepted after manual inspection of its corresponding MS/MS spectra, isotopic distribution, and its consistent retention time despite being above the 5% FDR threshold. Quantification was performed within Skyline () and the results were exported for further visualization and analysis using the R programming language. Proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD003983. [...] Oxyester-linked 15N E2-O-Ub conjugate (UbcH5c(Ser22Arg/Cys85Ser)-O-Ub was generated as previously described (). Two-dimensional 1H-15N HSQC-TROSY experiments were performed with 200 μM 15N E2-O-Ub conjugate in 25 mM sodium phosphate, pH 7.0 and 150 mM NaCl on a Bruker 500 MHz AVANCE II NMR spectrometer. MBP-UHRF1 and/or HeDNA was added to experiments to a final concentration of 18 μM and 22 μM, respectively. NMR data was processed with NMRPipe () and peak intensities were determined using NMRViewJ () (OneMoonScientific). Relative peak intensity changes were determined as the absolute peak intensity divided by the initial intensity of the E2-O-Ub conjugate in the absence of additives. […]

Pipeline specifications

Software tools NMRPipe, NMRViewJ
Application NMR-based metabolomics