Shotgun proteomics

ollowing HA-immunoprecipitation of C9orf72 from SMCR8 KO cells, proteins were precipitated with 20% TCA and incubated for 20 min on ice. After centrifugation at 20,000 x g, 4°C for 30 min, the supernatant was discarded, 10% cold TCA was added to pellets and centrifuged again. Pellets were washed 3x in cold acetone, centrifuged and then dried in a speed vacuum concentrator. For in-solution tryptic digestion, pellets were resolved in 50 mM ammonium bicarbonate (ABC) pH 8.0 with 10% acetonitrile (ACN). 0.5 µg trypsin was added and proteins were digested for 4 h at 37°C. Reaction was stopped by adding 5% formic acid in 5% ACN for 10 min at RT. Peptides were dried and reconstituted in 1% trifluoroacetic acid (TFA) in 5% ACN. For in-gel tryptic digestion, eluted proteins were separated on a polyacrylamide gel and gel was cut in small gel pieces (1 x 1 mm). Gel pieces were washed 3x in 50 mM ABC with 50% ethanol for each 15 min at RT, dehydrated by incubating 2x in absolute ethanol and dried using the speed vacuum concentrator. Proteins were reduced by incubating gel pieces in 10 mM DTT in 50 mM ABC for 45 min at 55°C and then alkylated by adding 55 mM iodoacetamide in 50 mM ABC for 30 min at RT. Gel pieces were washed in 50 mM ABC for 15 min at RT and dehydrated in absolute ethanol for 15 min at RT twice in turns. Following a last step of dehydration with ethanol, gel pieces were dried completely. 0.5 µg trypsin in 50 mM ABC was incubated on gel pieces overnight at 37°C. Peptides were extracted by incubating gel pieces with 3% TFA in 30% ACN, 2x 70% ACN and 2x 100% ACN. All elution steps were conducted for 20 min at RT and eluates were collected after every step. Peptide eluates were evaporated to approximately 1/5 of the original volume and mixed 1:1 with 1% TFA in 5% ACN. Desalting of peptides was accomplished on custom-made C18 stage tips and peptides were reconstituted in 0.1% formic acid for MS analysis. Following reconstitution in 0.1% formic acid, samples were loaded on a 75 µm x 15 cm fused silica capillary (custom-made) packed with C18Q resin (Reprosil-PUR 120, 1.9 μm, Dr. Maisch) and separated using EASY nLC 1200 liquid chromatography (Thermo Scientific). For the C9orf72 interactome, peptides were separated using a 35 min ACN gradient in 0.1% formic acid at a flowrate of 400 nL/min (10-38% ACN for 23 min, 38-60% ACN for 2 min and 60-100% ACN for 5 min). For the C9orf72 ubiquitination status, peptide separation was performed with a 60 min ACN gradient in 0.1% formic acid at a flowrate of 400 nL/min (10-38% ACN for 35 min, 38-60% ACN for 5 min and 60-100% ACN for 10 min). Separated peptides were detected using Q Exactive HF mass spectrometer (Thermo Scientific).