Project description:Fertilized eggs (wild type or ikke-1 mutant) were collected from approximately 10,000 or 70,000 adult hermaphrodites, equivalent to 5 or 35 plates of 10-cm dish culture, at 60–72 h after the L1 stage using the bleaching method, and immediately frozen in liquid nitrogen. The eggs were thawed on ice in HEPES-RIPA3 buffer (20 mM HEPES-KOH pH 7.4, 150 mM NaCl, 1% [v/v] NP40, 0.25% [w/v] Na-deoxycholate, 0.05% [w/v] SDS, and 50 mM NaF) with Complete EDTA-free Protease Inhibitor Cocktail (Roche, Basel, Switzerland) and PhosSTOP (Roche). The eggs were sonicated, incubated for 15 min at 4°C, and centrifuged at 16,200 ×g for 15 min at 4°C. The supernatant was collected and centrifuged at 16,200 ×g for 10 min at 4°C. The supernatant was transferred to a fresh tube, and GFP-Trap magnetic agarose beads (Proteintech, Rosemont, IL, USA) that had been prewashed with HEPES-RIPA3 buffer were added. The beads and supernatant were rotated for 2 h at 4°C. After rotation, the beads were transferred to a Protein LoBind tube (Eppendorf, Hamburg, Germany), washed three times with HEPES-RIPA3 buffer, and washed twice with 50 mM ammonium bicarbonate. The beads were divided to three new Protein LoBind tube with 50 µL of 50 mM ammonium bicarbonate. The proteins on the beads were digested using three methods. A) Beads and 200 ng of trypsin/LysC mix (Promega) were mixed and shaken overnight at 37°C. B) Beads and 200 ng of chymotrypsin (Roche) were mixed and shaken with 10 mM CaCl2 overnight at 25°C. C) Beads and 40 ng of Asp-N (Promega) were mixed and shaken overnight at 37°C. After the reaction, supernatants containing the digested peptides were collected, reduced, alkylated, acidified to pH 2.5 with TFA, and desalted using GL-Tip SDB (GL Sciences, Tokyo, Japan). The eluates were evaporated and dissolved in 3% ACN and 0.1% TFA. LC-MS/MS analysis of the resulting peptides was performed on an EASY-nLC 1200 UHPLC system connected to an Orbitrap Fusion mass spectrometer using a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on a C18 reversed-phase column with a linear 4–32% ACN gradient for 0–100 min, followed by an increase to 80% ACN for 10 min, which was held at 80% ACN for 10 min. The mass spectrometer was operated in data-dependent acquisition mode with a maximum duty cycle of 3 s. The MS1 spectra were measured at a resolution of 60,000, an AGC target of 4e5, and a mass range of 350–1500 m/z. HCD MS/MS spectra were acquired using an Orbitrap with a resolution of 30,000, AGC target of 5e4, isolation window of 1.6 m/z, maximum injection time of 54 ms, and normalized collision energy of 30. Dynamic exclusion was set to 15 s. Raw data were directly analyzed against the C. elegans WormBase protein database supplemented with the EPG-7-GFP sequence using Proteome Discoverer 2.4 (Thermo Fisher Scientific) with the Sequest HT search engine. The search parameters were as follows: (a) trypsin, chymotrypsin, or Asp-N as an enzyme with up to two, three, or three missed cleavages, respectively; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.02 Da; (d) carbamidomethylation of cysteine as a fixed modification; (e) acetylation of the protein N-terminus, oxidation of methionine, and phosphorylation of serine, threonine, and tyrosine as variable modifications. Peptides were filtered at a false discovery rate of 1% using Percolator node. Label-free quantification was performed based on the intensities of the precursor ions using a precursor-ion quantifier node.
2024-02-01 | PXD048129 | JPOST Repository
