Computational protocol: Interaction of CK1δ with γTuSC ensures proper microtubule assembly and spindle positioning

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Protocol publication

[…] Hrr25-TAP was purified from yeast cells expressing the tagged protein from the endogenous genomic locus as described (), with the modification that 150 mM KCl was used throughout the purification.Hrr25-TAP and associated proteins were incubated in digestion buffer (8 M urea, 100 mM Tris, pH 8.5). The mixture was brought to 5 mM Tris (2-carboxyethyl) phosphine and incubated at room temperature for 15 min. Iodoacetamide was then added to 10 mM and the resultant mixture incubated at room temperature for 20 min in dark. The samples were then diluted fourfold with 100 mM Tris (pH 8.5) and digested overnight with 1/40 enzyme/protein ratio of trypsin (Promega, Madison, WI) at 37°C.Digested peptide mixtures were pressure loaded onto a Kasil-fritted fused silica capillary column (250-μm inner diameter [i.d.]) packed with 3 cm of 5-μm Partisphere strong cation exchange resins (SCX; Whatman, Clifton, NJ) and 3 cm of 5-μm Aqua C18 resins (RP; Phenomenex, Ventura, CA). The column was then washed with buffer containing 95% water, 5% acetonitrile, and 0.1% formic acid. After desalting, this sample-loaded back-end column was then connected to a 100-μm-i.d. capillary column with a 5-μm pulled tip packed with 10 cm of 3-μm Aqua C18 material through a zero-dead-volume union (Upchurch, Oak Harbor, WA), and the entire three-phase column was placed inline with an Agilent 1200 quaternary HPLC (Agilent, Palo Alto, CA), and a modified 9-step MudPIT analysis described previously () was performed. Three buffer solutions were used: 5% acetonitrile/0.1% formic acid (buffer A); 80% acetonitrile/0.1% formic acid (buffer B), and 500 mM ammonium acetate/5% acetonitrile/0.1% formic acid (buffer C). The first step consisted of a 60-min gradient from 0 to 100% buffer B. Steps 2–9 had the following gradient profile: 3 min of 100% buffer A, 5 min of X% buffer C (X = 10, 20, 30, 40, 50, 60, 80, and 100%, respectively, for the analysis of steps 2–9), a 10-min gradient from 0 to 10% buffer B, a 70-min gradient from 10 to 45% buffer B, a 10-min gradient from 45 to 100% buffer B, and a 10-min equilibration of 100% buffer A. As peptides were eluted from the microcapillary column, they were electrosprayed directly into an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) with the application of a distal 2.5-kV spray voltage. A cycle of one full-scan mass spectrum (400–1600 m/z, 60,000 resolution), followed by 10 data-dependent collision-induced dissociation tandem mass spectrometry (MS/MS) spectra at a 35% normalized collision energy, was repeated continuously throughout each step of the multidimensional separation. Application of mass spectrometer scan functions and HPLC solvent gradients was controlled by the Xcalibur data system (Thermo Fisher Scientific).MS/MS spectra were extracted using RawXtract (version 1.9.9; ) and searched with the ProLuCID algorithm () against a Saccharomyces cerevisiae database concatenated to a decoy database in which the sequence for each entry in the original database was reversed. A static modification (+57.02146) on cysteine was added to the search. The precursor mass tolerance was set as 50 ppm, and fragment mass tolerance was set as 600 ppm. The enzyme specificity was semitryptic, with number of missed cleavages at 2. ProLuCID search results were assembled and filtered using the DTASelect, version 2.0, program (), requiring a minimum of two peptides per protein identification. The protein identification false-positive rate was held <1%, and all peptide-spectra matches were required to have <5 ppm mass error. […]

Pipeline specifications

Software tools RawConverter, ProLuCID, DTASelect
Application MS-based untargeted proteomics
Organisms Saccharomyces cerevisiae, Homo sapiens