Recombinant proteins and general reagents
Protein kinase assays
Cloning and recombinant protein purification from E. coli
Differential scanning fluorimetry
Human cell culture and cell treatments
Human cell lysis, immunoprecipitation, and Western blot analysis
Detection of sulfenylated and glutathionylated proteins by immunoblotting
Aurora A sample preparation for MS analysis of intramolecular disulfide bond formation
MS data analysis
Identification, alignment, and visualization of protein kinase–related sequences
Yeast strains, plasmids, and growth conditions
Analysis of S. pombe proteins by immunoblotting
Analysis of S. pombe cell length at division and CDC25-GFP localization
Assessing growth and salt stress sensitivity of S. pombe
Statistical analysis
Peptides were separated on a Ultimate 3000 nano system (Dionex) by reverse-phase high-performance liquid chromatography, using a trapping column (PepMap 100, C18, 300 μm by 5 mm) equilibrated in loading buffer [3% (v/v) acetonitrile and 0.1% (v/v) TFA] at a flow rate of 9 μl/min for 7 min. Chromatographic separation was performed using an EASY-Spray C18 column (75 μm by 500 mm, 2-μm bead diameter) at a flow rate of 0.3 μl/min over a 30-min gradient from 3% buffer A [0.1% (v/v) formic acid]:97% buffer B [80% (v/v) acetonitrile and 0.1% (v/v) formic acid] to 20% buffer A:80% buffer B. Data were acquired using a Thermo Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific). All spectra were acquired in the Orbitrap in a data-dependent analysis mode using a top speed approach (3-s cycle time), with ions being subjected to higher-energy collisional dissociation (HCD) (normalized collision energy of 32%). MS1 parameters: 60K resolution at 200 mass/charge ratio (m/z); AGC, 4 × 105; maximum injection time, 50 ms; mass range, 350 to 2000; charge stated, 2+ to 6+. MS2 parameters: 30K resolution at 200 m/z; AGC, 5 × 104; maximum injection time, 54 ms. A dynamic exclusion window was applied for 60 s at a tolerance of 10 parts per million (ppm).