APEX-IP for LC-MS/MS

From strains validated to express active APEX:

• Prepare pools of elutriated cells, collected at the desired cell size, resuspended in freezing buffer (15% glycerol, 150 mM potassium acetate, 2 mM magnesium acetate, 20 mM HEPES/sodium hydroxide pH 7.2, 0.5% (^w/v) glucose, 100 μg/mL cycloheximide), and stored at -80 °C.

• For each labeling reaction (pool), thaw 1E+09 cells on ice, and wash in 10 mL 0.1M MES/sodium hydroxide pH 6.5.

• Resuspend in 2 mL of this buffer containing 0.01% digitonin (10 μL added from a 20 mg/mL digitonin stock in DMSO, stored at -80 °C).

• Incubate in a 30 °C shaking water bath for 30 min.

• Collect cells by centrifugation, and wash with 10 mL of ice-cold 1.2M sorbitol/PBS.

• Resuspend in 2 mL of 1.2M sorbitol/PBS containing 2.5 mM phenol-biotin (10 μL added from a 0.5M stock in DMSO, stored at -80 °C), 20 μL from a 20 U/μL stock of SUPERase•In RNase Inhibitor, 20 μL from a 10 mg/mL stock of cycloheximide, and protease inhibitors.

• Incubate on ice for 90 min.

• About 15 min before the end of the 90 min incubation on ice, prepare a quenching solution by mixing the following: 200 μL Trolox (from a 0.5M stock in DMSO, stored at -80 °C); 200 μL sodium azide (from a 1M stock in water, stored at -80 °C); 2 mL of a 10 mM sodium ascorbate solution in PBS, prepared fresh.

• Also before the end of the 90 min incubation on ice, prepare a 0.2M stock of hydrogen peroxide (by dilution of the 30% (9.8M) hydrogen peroxide solution; stored at 4 °C).

• At the end of the 90 min incubation on ice, expose the cells to 2mM hydrogen peroxide (20 μL were added to the 2 mL cell suspension from the 0.2M solution).

• Vortex briefly, and incubate on ice for 2 min.

• Stop labeling by adding 2 mL of the freshly prepared quenching solution described above.

• Collect cells by centrifugation and wash with 10 mL of TBS, pH 7.5.

• Resuspend the cells in 3 mL of TBS, pH 7.5.

• Add 1.5 mL of glass beads to each tube.

• Break the cells with 6 cycles of 30 sec vortexing-30 sec on ice.

• Centrifuge for 10 min in the cold.

• Transfer the supernatant in 15 mL screw-cap tubes.

• Add 0.2 mL of Myone.C1 streptavidin beads. Incubate with a rotisserie mixer for 1 h at room temperature.

• Use the magnetic rack to isolate the beads. Remove the supernatant. Wash two times with 10 mL TBS, 2 M urea, pH 7.5.

• Wash with 10 ml 0.1 M ammonium carbonate pH 7.7.

• Resuspend in 0.5 ml 0.1 M ammonium carbonate pH 7.7.