Five micrograms of Aurora A purified in the absence of DTT was heated at 80°C in 0.06% (w/v) RapiGest SF (Waters) dissolved in 25 mM ammonium bicarbonate for 10 min. Samples were digested overnight at 25°C using a 20:1 (w/w) ratio of Aurora A:Chymotrypsin (Promega), with shaking at 600 rpm. The sample was equally split into two, one aliquot of which was reduced with DTT (3 mM) at 60°C for 10 min, cooled, and alkylated with 10 mM iodoacetamide (IAA) at room temperature for 30 min in the dark. Excess IAA was quenched by addition of DTT to a final concentration of 7 mM. The second sample was left on ice. RapiGest hydrolysis was induced in both samples by the addition of 1.5% (v/v) trifluoroacetic acid (TFA) and 3% (v/v) acetonitrile, shaking at 600 rpm and 37°C for 2 hours. Insoluble products were removed by centrifugation (13,000g for 15 min at 4°C). Samples were subjected to strong cation exchange using in-house packed stage tips (Empore Supelco 47-mm Cation Exchange disk #2251), three disks per 200 μl of tip. All centrifugation steps were performed at 2000g for 3 min until all liquid had passed through the stage tip. Briefly, tips were equilibrated with sequential washes in acetone, methanol, water, 5% (v/v) ammonium hydroxide, and water (2 × 200 μl of each). Peptide samples were thrice passed through the equilibrated stage tip and washed with 250 μl of 1.5% (v/v) TFA, before elution with 250 μl of 5% (v/v) ammonium hydroxide. Eluted material was dried using cooled vacuum centrifugation. Peptides were solubilized in 20 μl of 3% (v/v) acetonitrile and 0.1% (v/v) TFA and sonicated for 10 min before centrifugation (13,000g for 15 min at 4°C) to remove insoluble material before LC-MS/MS analysis.

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