PXD065081-1

PXD065081 is an original dataset announced via ProteomeXchange.

Title	TMTpro-16plex analysis of worm embryos with unc-32 RNAi
Description	An rrf-1(pk1417) mutant strain was used to knockdown unc-32 gene function specifically in the germline, in which somatic RNAi, but not germline RNAi, was significantly repressed. Gravid adult hermaphrodites were collected in M9 buffer and bleached to obtain hard-shelled embryos. The embryos were washed with M9 buffer three times and lysed in Gdm-TCEP buffer (8 M guanidine-HCl, 100 mM HEPES-NaOH pH7.5, 10 mM TCEP, and 40 mM CAA). After heating and sonication, proteins (100 µg each) were purified by methanol-chloroform precipitation and resuspended in 20 µL of 0.1% RapiGest SF (Waters) in 50 mM triethylammonium bicarbonate. After a second round of sonication and heating, the proteins were digested with 1 µg of trypsin/Lys-C mix (Promega) for 16 h at 37 ºC. Peptide concentrations were determined using the Pierce quantitative colorimetric peptide assay. Approximately 40 µg of peptides per sample were labeled with 125 µg of TMTpro-16plex reagents (Thermo Fisher Scientific) for 1 h at 25 ºC. The reaction was quenched with hydroxylamine, after which all TMT-labeled samples were pooled, acidified with TFA, and fractionated by offline high-pH reversed-phase chromatography using a Vanquish DUO UHPLC system. Peptides were separated into 48 fractions and then consolidated into 16 fractions. Each fraction was evaporated using a SpeedVac concentrator and dissolved in 3% ACN and 0.1% TFA. LC-MS/MS analysis of the resulting peptides (500 ng per fraction) 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). Peptides were separated on a 75 µm inner diameter x 150 mm C18 reversed-phase column (Nikkyo Technos) with a linear gradient of 4–20% ACN for 0–115 min and 20–32% ACN for 115–160 min, followed by a ramp to 80% ACN for 10 min and a final hold at 80% ACN for 10 min. The mass spectrometer was operated in the data-dependent acquisition mode using the top 10 MS/MS method. The MS1 spectra were measured at a resolution of 70,000, an 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, an AGC target of 1e5, isolation window of 0.7 m/z, maximum injection time of 150 ms, and normalized collision energy of 32. Dynamic exclusion was set to 20 s. Raw data were directly analyzed against the C. elegans WormBase protein database (release WS285) using Proteome Discoverer version 2.5 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) TMTpro of lysine and peptide N-terminus, and carbamidomethylation of cysteine as fixed modifications; and (e) oxidation of methionine as a variable modification. Peptides were filtered at a false discovery rate of 1% using a percolator node. Normalization was performed such that the total sum of the abundance values for each TMT channel for all peptides was the same.
HostingRepository	jPOST
AnnounceDate	2026-04-09
AnnouncementXML	Submission_2026-04-08_19:48:56.543.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Hidetaka Kosako
SpeciesList	scientific name: Caenorhabditis elegans; NCBI TaxID: 6239;
ModificationList	S-carboxamidomethyl-L-cysteine; L-methionine sulfoxide; unknown modification; unknown modification
Instrument	Q Exactive Plus

Revision	Datetime	Status	ChangeLog Entry
0	2025-06-16 21:11:33	ID requested
⏵ 1	2026-04-08 19:48:57	announced

Dataset with its publication pending

submitter keyword: TMT, C. elegans, unc-32

Hidetaka Kosako
lab head
Hidetaka Kosako
contact affiliation	Tokushima University
dataset submitter

jPOST dataset URI

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