Affinity Purification and Mass Spectrometry of FLAG-Tagged Proteins

Yeast cells were grown in 1.2 L YPD media until an OD₆₀₀ of 1.0, washed with PBS and flash frozen in liquid nitrogen. Thawed cell pellets were resuspended in IP buffer (40 mM HEPES pH7.5, 150 mM NaCl, 10% glycerol, 0.1% Tween-20) and lysed with glass beads using a Biospec bead beater. The lysate was centrifuged at 45,000 rpm at 4 °C for 1.5 h and the supernatant was incubated with anti-FLAG agarose beads at 4 °C for 4 h. The beads were washed extensively in IP buffer. Proteins were disulphide-reduced by 25 mM DTT at 37 °C for 40 min, alkylated by adding 50 mM iodoacetamide, and then digested with sequencing-grade trypsin (Promega) at 37 °C overnight. The supernatant was desalted using C18 solid-phase cartridges and lyophilized. The dried peptides were reconstituted in 0.1% FA and loaded onto an Acclaim PepMap 100 C18 LC column (Thermo Fisher) utilizing a Thermo Easy nLC 1000 LC system (Thermo Fisher) connected to Q Exactive HF mass spectrometer (Thermo Fisher) and data were analyzed as described^{68 (link)}.