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Multivalent Coiled-Coil Interactions as a Conserved Design Principle for Centrosome Assembly

ABSTRACT: Crosslinking reactions were prepared at room temperature with 1uM dephosphorylated SPD-5, 1uM Kinase Dead (KD) PLK-1 , 0.2 mM ATP, 10 mM MgCl2, 150mM KCl, 25mM HEPES, pH7.4 and 0.5mM DTT. Samples were incubated for 2 hr at room temperature followed by addition of 1.47uL of DMTMM (20mg/mL) for 45min at room temperature (shaking at 300 rpm). To quench the reaction, we added 2.56uL of Ammonium Bicarbonate (50mM) for 15min at room temperature (shaking at 300rpm).

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Caenorhabditis Elegans (ncbitaxon:6239)

SUBMITTER: Jeffrey Woodruff

PROVIDER: MSV000094199 | MassIVE | Wed Feb 28 17:05:00 GMT 2024

REPOSITORIES: MassIVE

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Project description:Sample preparation: P-AMPK+AMP, P-AMPK+ATPγS, and AMPK+ATPγS protein samples were crosslinked in HBST buffer (25 mM HEPES pH 7.5 at room temperature, 200 mM NaCL, and 1 mM TCEP). Non-crosslinked negative controls were generated using the same procedure without the addition of DSSO. Reactions were initiated by spiking 1 uL of 75 mM DSSO (Thermo-Fisher) in DMSO into a 50 uL solution containing 10 uM protein and mixing. The final molar ratio of crosslinker:protein was 150:1. The reactions were incubated at 25°C for 45 minutes prior to being quenched by the addition of Tris pH 8.0 to a final concentration of 50 mM. Each crosslink reaction was done in triplicate and the reaction replicates were pooled after quenching. 10 μL samples of the pooled samples were loaded for SDS-PAGE analysis with Coomassie staining to confirm the presence of crosslinked protein. The remaining 140 μL of crosslinked and non-crosslinked samples were then acetone precipitated at -20°C overnight and the protein was pelleted by centrifugation at 16,000 rcf for 5 minutes at 4°C. After decanting, the pellets were dried for 15 minutes at room temperature before being resuspended in 12.5 uL of resuspension buffer (50 mM ammonium bicarbonate, 8M urea, pH 8.0). ProteaseMAX (Promega) was added to 0.02% and the solutions were mixed on an orbital shaker operating at 400 RPM for 5 minutes. After resuspension, 87.5 uL of digestion buffer (50 mM ammonium bicarbonate, pH 8.0) was added. Cysteines in the protein samples were reduced and akylated as follows. DTT was added to 5 mM final concentration and the protein solutions were incubated at 56°C in an orbital shaker operating at 400 RPM and for 20 minutes. After reduction, 2.7 uL of 550 mM iodoacetamide was added and the solutions were incubated in the dark at room temperature for 15 minutes. Reduced and alkylated protein solutions were digested using trypsin at a ratio of 1:200 (w/w trypsin:protein) and incubating at 37°C. After overnight incubation at 37°C, the samples were acidified by the addition of trifluoroacetic acid (TFA, Thermo-Fischer) to 1%. Samples were then frozen and stored at -20°C until analysis.

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Multivalent Coiled-Coil Interactions as a Conserved Design Principle for Centrosome Assembly

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