Ltq orbitrap fusion

Immuno-purified FOXK1 protein was subjected to SDS-PAGE and protein bands were stained with Coomassie brilliant blue. Following gel-extraction, reduction of samples was performed by adding 5 mM DTT in 50 mM ammonium bicarbonate. Alkylation was performed with 50 mM chloroacetamide and 50 mM ammonium bicarbonate. Trypsin digestion was performed for 8 hours at 37°C. Samples were loaded and separated on a homemade reversed-phase column (150 µm i.d. x 150 mm) with a 106-min gradient from 0–40% acetonitrile (0.2% FA) and a 600 nl/min flow rate on an Easy nLC-1,000 (Thermo Fisher Scientific) connected to an LTQ-Orbitrap Fusion (Thermo Fisher Scientific). Each full MS spectrum acquired with a 70,000 resolution was followed by 10 MS/MS spectra, where the 10 most abundant multiply charged ions were selected for MS/MS sequencing. Tandem MS experiments were performed using high-energy C-trap dissociation (HCD) and electron transfer dissociation (ETD) acquired in the Orbitrap. Peaks were identified using a Peaks 7.0 (Bioinformatics Solution Inc.) and peptide sequences were blasted against the human Uniprot database (74,530 sequences). Tolerance was set at 10 ppm for precursor and 0.01 Da for fragment ions during data processing and with carbamidomethylation (C), oxidation (M), deamidation (NQ), and Hex-N-acylation (ST) as variable modifications.