Computational protocol: Insights from the reconstitution of the divergent outer kinetochore of Drosophila melanogaster

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Protocol publication

[…] About 0.8 mg ml−1 DmMis12a was mixed with 0.6 mM BS2G-H6/D6 (Creative Molecules, in a final volume of 50 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 cross-linked 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 []. False discovery rates (FDRs) were estimated using xProphet [], and results were filtered according to the following parameters: FDR < 0.05, min delta score = 0.90, MS1 tolerance window of −4 to 4 ppm, Id-score > 22. Cross-links were visualized using the xVis web server []. […]

Pipeline specifications

Software tools xQuest, xVis
Application MS-based untargeted proteomics
Organisms Drosophila melanogaster, Homo sapiens