DOI: 10.25345/c5qn5zr2w
title: MassIVE MSV000100277 - Crosstalk of histone H3pT11 and H3K4me3 via interactions of NUAK1 with the SETD1A complex regulates glucose metabolism
title: TCA precipitated protein samples were analyzed independently by Multidimensional Protein Identification Technology (MudPIT). Briefly, precipitated protein samples were resuspended in 100mM Tris pH 8.5, 8M urea to denature the proteins. Proteins were reduced and alkylated prior to digestion with recombinant LysC (Promega) and trypsin (Promega). Reactions were quenched by the addition of formic acid (FA) to a final concentration of 5%. Peptide samples were pressure-loaded onto 100um fused silica microcapillary columns packed first with 9cm of reverse phase material (Aqua; Phenomenex), followed by 3cm of 5um Strong Cation Exchange material (Luna; Phenomenex), followed by 1cm of 5um C18 RP. The loaded microcapillary columns were placed in-line with a 1200 G1311 Quartenary HPLC (Agilent). The application of a 2.5kV distal voltage electrosprayed the eluting peptides directly into orbitrap Elite mass spectrometers (Thermo Scientific) equipped with a custom-made nano-LC electrospray ionization source. Full MS spectra were recorded on the eluting peptides over a 400 to 1600mz range, followed by fragmentation in the ion trap (at 35% collision energy) on the first to fifth most intense ions selected from the full MS spectrum. Dynamic exclusion was enabled for 90s. Mass spectrometer scan functions and HPLC solvent gradients were controlled by the XCalibur data system (Thermo Scientific). RAW files were extracted into .ms2 file format using RawDistiller v. 1.0, in-house developed software. RawDistiller settings were used to abstract MS1 scan profiles by Gaussian fitting and to implement dynamic offline lock mass using six background polydimethylcyclosiloxane ions as internal calibrants. MS2 spectra were first searched using ProLuCID with a mass tolerance of 10ppm for peptide and fragment ions. Trypsin specificity was imposed on both ends of candidate peptides during the search against a protein database combining 81,592 human proteins (NCBI 20201123 release), as well as 426 common contaminants such as human keratins, IgGs, and proteolytic enzymes. To estimate false discovery rates (FDR), each protein sequence was randomized (keeping the same amino acid composition and length) and the resulting shuffled sequences were added to the database, for a total search space of 163,860 amino acid sequences. A mass of 57.0125Da was added as a static modification to cysteine residues and 15.9949Da was differentially added to methionine residues. DTASelect v.1.9 was used to select and sort peptide/spectrum matches (PSMs) passing the following criteria set: PSMs were only retained if they had a DeltCn of at least 0.08; minimum XCorr values of 1.8 for singly, 2.1 for doubly, and 2.5 for triply charged spectra,peptides had to be at least 7 amino acids long. Results from each sample were merged and compared using CONTRAST. Combining all replicate runs, proteins had to be detected by at least 2 peptides and/or 2 spectral counts. Proteins that were subsets of others were removed using the parsimony option in DTASelect on the proteins detected after merging all runs. Proteins that were identified by the same set of peptides (including at least one peptide unique to such protein group to distinguish between isoforms) were grouped together, and one accession number was arbitrarily considered as representative of each protein group. NSAF7 was used to create the final reports on all detected peptides and non-redundant proteins identified across the different run.