LFQ of GFP-Trap IP-MS from BDKO cells expressing Tmem63b-GFP

Cell lysates were prepared from Tmem63b-GFP-expressing BDKO cells and incubated with the GFP-Trap magnetic agarose beads (Proteintech). After rotation at 4˚C for 3 hr, the beads were precipitated with the magnet, followed by washing several times using wash buffer containing 25 mM Tris-HCl (pH 8.0), 100 mM 6-aminocaprotic acid, 140 mM NaCl, 0.01% LMNG / 0.001% CHS, and applied to two additional washes with 50 mM ammonium bicarbonate. Subsequently, proteins bound to the beads were digested by adding trypsin/Lys-C mix (Promega) at 37°C for 16 hours, after which the resulting digested products were used for a series of steps including reduction, alkylation, acidification, and desalting using GL-Tip SDB (GL Sciences). The eluates were concentrated in a SpeedVac concentrator, followed by dissolution in a solution consisting of 0.1% trifluoroacetic acid and 3% acetonitrile (ACN). LC-MS/MS analysis of the generated peptides was executed on an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer via a nanoelectrospray ion source (Thermo Fisher Scientific). The separation of peptides occurred on a 75 μm inner diameter × 150 mm C18 reversed-phase column (Nikkyo Technos), using a linear 4–32% ACN gradient over 0–100 minutes, followed by a 10-minute increase to 80% ACN. The mass spectrometer was operated in a data-dependent acquisition mode, with a maximum duty cycle of 3 seconds. MS1 spectra were measured with a resolution of 120,000, an automatic gain control (AGC) target of 4e5, and a mass range from 375 to 1,500 m/z. HCD MS/MS spectra were acquired in the linear ion trap with an AGC target of 1e4, an isolation window of 1.6 m/z, a maximum injection time of 100 msec, and a normalized collision energy of 30. Dynamic exclusion was set to 20 seconds. The raw data were directly analyzed against the SwissProt database restricted to Mus musculus using Proteome Discoverer version 2.5 (Thermo Fisher Scientific) with the Sequest HT search engine. The search parameters included trypsin as the enzyme with up to two missed cleavages, a precursor mass tolerance of 10 ppm, a fragment mass tolerance of 0.6 Da, carbamidomethylation of cysteine as a fixed modification, and acetylation of the protein N-terminus and oxidation of methionine as variable modifications. Peptides were filtered at a false-discovery rate of 1% using the percolator node. Label-free precursor ion quantification was conducted using the precursor ions quantifier node, and normalization was performed to ensure that the total sum of abundance values for each sample over all peptides remained consistent.