LFQ of immunoprecipitates with GFP-Trap from GFP-, OfMasc-GFP-, or Del1-OfMasc-GFP-transfected cells

OfMasc-GFP derivatives or GFP were transiently expressed in OfTC cells. At 2 days post transfection, OfTC cells were treated with 100 nM Bortezomib for 1 day. The OfT1C cells were then fixed with 0.1% formaldehyde, and the fixation was quenched with 300 mM glycine. The cells were lysed on ice in 1 mL of RIPA buffer. After centrifugation, the supernatants were incubated with GFP-Trap Magnetic Agarose (ChromoTek) for 3 h at 4ºC. Proteins bound to the beads were digested by adding 200 ng of Trypsin/Lys-C mix (Promega) for 16 h at 37ºC. The digests were reduced, alkylated, acidified with trifluoroacetic acid, and desalted using a GL-Tip SDB (GL Sciences). LC-MS/MS analysis of the resultant peptides was performed using an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer equipped with a nanoelectrospray ion source (Thermo Fisher Scientific). Raw data were directly analyzed against the wFur encoded protein data predicted from an in-house assembled wFur draft genomic sequence and O. furnacalis protein data ‘GCF_004193835.1_ASM419383v1_protein.faa’ downloaded from NCBI supplemented with OfMasc-GFP and GFP-Trap sequences using Proteome Discoverer version 2.4 (Thermo Fisher Scientific) with Sequest HT search engine. Label-free precursor ion quantification was performed using the precursor ions quantifier node.