Computational protocol: USP9X regulates centrosome duplication and promotes breast carcinogenesis

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Protocol publication

[…] To identify proteins associated with FLAG-USP9X or CEP131, LC-MS/MS analysis was performed using a Thermo Finnigan LTQ linear ion trap mass spectrometer in line with a Thermo Finnigan Surveyor MS Pump Plus HPLC system. Tryptic peptides generated were loaded onto a trap column (300SB-C18, 5 × 0.3 mm, 5 μm particle; Agilent Technologies, Santa Clara, CA), which was connected through a zero dead volume union to the self-packed analytical column (C18, 100 μm i.d × 100 mm, 3 μm particle; SunChrom, Germany). The peptides were then eluted over a gradient (0–45% B in 55 min, 45–100% B in 10 min, where B=80% acetonitrile, 0.1% formic acid) at a flow rate of 500 nl min−1 and introduced online into the linear ion trap mass spectrometer (Thermo Fisher Corporation, San Jose, CA) using nano electrospray ionization. Data-dependent scanning was incorporated to select the five most abundant ions (one microscan per spectra; precursor isolation width 1.0 m/z, 35% collision energy, 30 ms ion activation, exclusion duration: 90 s; repeat count: 1) from a full-scan mass spectrum for fragmentation by collision induced dissociation. MS data were analysed using SEQUEST (v. 28) against NCBI human protein database (14 December 2011 downloaded, 33,256 entries), and results were filtered, sorted and displayed using the Bioworks 3.2. Peptides (individual spectra) with Preliminary Score (Sp)≥500; Rank of Sp (RSp)≤5; and peptides with +1, +2 or +3 charge states were accepted if they were fully enzymatic and had a cross-correlation (Xcorr) of 1.90, >2.75 and >3.50, respectively. The following residue modifications were allowed in the search: carbamidomethylation on cysteine as fix modification and oxidation on methionine as variable modification. Peptide sequences were searched using trypsin specificity, allowing a maximum of two missed cleavages. Sequest was searched with a peptide tolerance of 3.0 Da and a fragment ion tolerance of 1.0 Da. [...] The tryptic peptides of CEP131-conjugated ubiquitin were dissolved in 0.1% formic acid (FA), directly loaded onto a reversed-phase pre-column (Acclaim PepMap 100, Thermo Scientific). Peptide separation was performed using a reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific). The gradient comprises an increase from 2 to 35% solvent B (0.1% FA in 98% acetonitrile (ACN)) over 12 min and climbing to 80% in 4 min and then holding at 80% for the last 4 min, all at a constant flow rate of 400 nl min−1 on an EASY-nLC 1000 UPLC system. The resulting peptides were analysed by Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). The peptides were subjected to NSI source followed by MS/MS in Q Exactive (Thermo) coupled online to the UPLC. Intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were selected for MS/MS using NCE setting as 28; ion fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions above a threshold ion count of 5E3 in the MS survey scan with 10.0 s dynamic exclusion. The electrospray voltage applied was 2.0 kV. Automatic gain control was used to prevent overfilling of the Orbitrap; 5E4 ions were accumulated for generation of MS/MS spectra. For MS scans, the m/z scan range was 350–1,800. Fixed first mass was set at 100 m/z. The resulting MS/MS data were processed using the Mascot search engine (v.2.3.0). Tandem mass spectra were searched against CEP131 (Homo sapiens) database. Trypsin/P was specified as cleavage enzyme allowing up to four missing cleavages for CEP131. Mass error was set to 10 p.p.m. for precursor ions and 0.02 Da for fragment ions. Carbamidomethyl on Cys was specified as fixed modification and oxidation on Met; acetylation on Protein N-term were specified as variable modifications for CEP131. Specifically, ubiquitination on lysine was set as variable modification for CEP131. Peptide ion score was set to >20. Finally, two ubiquitination sites were identified in sample CEP131 (the protein coverage is 61.22%). All the detailed information was presented in . […]

Pipeline specifications

Software tools Comet, Mascot Server
Application MS-based untargeted proteomics