Dual-affinity purified proteins were separated by SDS-PAGE. The gels were fixed in 50% methanol/7% acetic acid for 1 h at RT, washed with water, stained with Coomassie Brilliant Blue (Thermo Fisher) for 2 h, and destained with water overnight. Each lane was then excised into seven pieces. Gel slices were washed in 50% acetonitrile and rehydrated with a 50 mM ammonia bicarbonate/trypsin solution and digested at 37 °C overnight. Peptides were then extracted with a series of elutions, dried in a speed vac, and solubilized in 0.1% formic acid in water for analysis by tandem mass spectrometry.
LC-MS/MS was performed on a LTQ Orbitrap Elite (Thermo Fisher) equipped with a Waters (Milford, MA, USA) NanoAcquity HPLC pump as described (13 (link)). Primary mass spectrometry data have been submitted to the MASSIVE database (MSV000086356) and PRIDE repository (PXD022135).
LC-MS/MS was performed on a LTQ Orbitrap Elite (Thermo Fisher) equipped with a Waters (Milford, MA, USA) NanoAcquity HPLC pump as described (13 (link)). Primary mass spectrometry data have been submitted to the MASSIVE database (MSV000086356) and PRIDE repository (PXD022135).