CuAAC Protocol and Sample Preparation for Proteomics Analysis.

Sample preparation was performed based on protocols from the Cravatt Laboratory (54). For SILAC samples, the heavy and light proteomes were combined in a 1:1 ratio before proceeding. CuAAC chemistry was performed as described above, using biotin azide instead of rhodamine azide. Excess reagents were removed using methanol/chloroform (MeOH/CHCl₃) precipitation: the samples were transferred to 15 mL conical tubes on ice, 2 mL cold MeOH was added, 0.5 mL cold CHCl₃ was added, and the sample was vortexed. Next, 1.0 mL cold PBS was added and the sample was vortexed and centrifuged at 5,000 rpm for 10 min. The top and bottom layers were removed, leaving the protein interface, and the sample was washed with 1:1 MeOH:CHCl₃ (1.0 mL, 3×). 2 mL of MeOH was added and the sample was sonicated with a probe sonicator, and then 0.5 mL CHCl₃ was added. Lastly, the sample was centrifuged at 5,000 rpm for 10 min to pellet protein and the supernatant was removed. Next, the samples were denatured and reduced: 500 µL 6M urea (in PBS, made fresh) and 50 µL premixed 100 mM TCEP/300 mM K₂CO₃ solution (in PBS) were added and the sample was sonicated and incubated for 30 min at 37 °C in shaker. Next, 70 µL 400 mM iodoacetic acid was added and incubated for 30 min at room temperature in the dark, 140 µL 10% SDS in PBS was added, and the sample was diluted with 5.5 mL PBS.