Computational protocol: Molecular requirements for the inter-subunit interaction and kinetochore recruitment of SKAP and Astrin

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Protocol publication

[…] 10 μM SKAP135–225 and 10 μM taxol-stabilized microtubules were mixed with DSS-H12/D12 (Creative Molecules, www.creativemolecules.com) in a weight ratio of 1:2.5 or with BS2G-H6/D6 (Creative Molecules) in a weight ratio of 1:1 in a final volume of 150 μl. After incubation for 30 min at 37 °C the reaction was quenched by adding 100 mM ammonium bicarbonate and incubating 15 min at 37 °C. Cross-linked proteins were digested and the peptides were enriched and analysed by liquid chromatography coupled to tandem mass spectrometry using a hybrid LTQ-Orbitrap Elite instrument (Thermo Fisher Scientific, Waltham, MA). Cross-links were identified by the dedicated software xQuest (ref. ). False discovery rates were estimated using xProphet (ref. ) and results were filtered according to the following parameters: false discovery rates=0.05, min delta score=0.90, MS1 tolerance window of −4 to 4 p.p.m., Id-score >22. Cross-links were visualized using the xVis web server. […]

Pipeline specifications

Software tools xQuest, xVis
Application MS-based untargeted proteomics