ABSTRACT: Fertilized eggs (wild-type, ikke-1, or allo-1 in four, three, or three biological replicates, respectively) were collected from approximately 10,000–20,000 adult hermaphrodites, equivalent to 4–7 plates of 10 cm dish culture, at 60–72 h after the L1 stage using the bleaching method. Eggs were suspended in 6 M guanidine-HCl, 100 mM Tris-HCl (pH 8.0), and 2 mM dithiothreitol and immediately frozen in liquid nitrogen. The thawed lysates were dissolved through heating and sonication, followed by centrifugation at 20,000 ×g for 15 min at 4°C. The supernatants were reduced in 5 mM dithiothreitol at room temperature for 30 min and alkylated in 27.5 mM iodoacetamide at room temperature for 30 min in the dark. Proteins (400 µg each) were purified using methanol-chloroform precipitation and solubilized with 50 µL of 0.1% RapiGest SF (Waters Corporation, Milford, MA, USA) in 50 mM triethylammonium bicarbonate. The proteins were digested with 4 µg of Trypsin/Lys-C mix (Promega) for 16 h at 37°C. Peptide concentrations were determined using a Pierce quantitative colorimetric peptide assay (Thermo Fisher Scientific). Approximately 150 µg of peptides for each sample was labeled with 0.2 mg of TMT-10plex reagents (Thermo Fisher Scientific) for 1 h at 25°C. After the reaction was quenched with hydroxylamine, all TMT-labeled samples were pooled, acidified with trifluoroacetic acid (TFA), and analyzed using a High-Select Fe-NTA phosphopeptide enrichment kit (Thermo Fisher Scientific). The eluates were acidified and fractionated using a Pierce High pH reversed-phase peptide fractionation kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Eight fractions were collected using 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, and 50% acetonitrile (ACN). Each fraction was evaporated using a SpeedVac concentrator and dissolved in 0.1% TFA. LC-MS/MS analysis of the resulting peptides was performed on an EASY-nLC 1200 UHPLC system connected to a Q Exactive Plus mass spectrometer using a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on a C18 reversed-phase column (75 µm × 150 mm; Nikkyo Technos, Tokyo, Japan) with a linear gradient of 4–20% ACN for 0–150 min and 20–32% ACN for 150–190 min, followed by an increase to 80% ACN for 10 min and a final hold at 80% ACN for 10 min. The mass spectrometer was operated in data-dependent acquisition mode using the top 10 MS/MS method. The MS1 spectra were measured at a resolution of 70,000, an automatic gain control (AGC) target of 3e6, and a mass range of 375-1400 m/z. HCD MS/MS spectra were acquired at a resolution of 35,000, AGC target of 1e5, isolation window of 0.7 m/z, maximum injection time of 100 ms, and normalized collision energy of 32. The dynamic exclusion was set at 30 s. Raw data were directly analyzed against the C. elegans WormBase protein database using Proteome Discoverer 2.1 (Thermo Fisher Scientific) with the Sequest HT search engine for identification and TMT quantification. The search parameters were as follows: (a) trypsin as an enzyme with up to two missed cleavages, (b) precursor mass tolerance of 10 ppm, (c) fragment mass tolerance of 0.02 Da; (d) TMT of lysine and peptide N-terminus and carbamidomethylation of cysteine as fixed modifications, and (e) oxidation of methionine, deamidation of asparagine and glutamine, and phosphorylation of serine, threonine, and tyrosine as variable modifications. Peptides were filtered at a false-discovery rate of 1% using a percolator node.
