Dataset Information

DIA-PASEF analysis of Shu immunoprecipitates from control and Shu knockdown Drosophila OSCs

ABSTRACT: After control or Shu knockdown (n = 3 each), OSCs were lysed in binding buffer [50 mM Tris-HCl (pH 8.0), 150 mM KOAc, 5 mM Mg(OAc)2, 5 mM DTT, 0.1% NP-40, 2 µg/mL leupeptin, 2 µg/mL pepstatin A, 0.5% aprotinin, 10% glycerol] and centrifuged to remove insoluble debris. The lysates were then incubated with anti-Shu antibody bound to Dynabeads Protein G (Thermo Fisher Scientific) at 4°C for 2 h. The beads were washed five times with binding buffer and twice with 50 mM ammonium bicarbonate. Proteins on the beads were digested by adding 400 ng of trypsin/Lys-C mix (Promega) at 37 °C overnight. The digests were reduced, alkylated, acidified, and desalted using GL-Tip SDB (GL Sciences). The eluates were evaporated in a SpeedVac concentrator and dissolved in 0.1% trifluoroacetic acid and 3% acetonitrile (ACN). LC-MS/MS analysis of the resultant peptides was performed on a nanoElute 2 coupled with a timsTOF HT mass spectrometer (Bruker). The peptides were separated on a 75-μm inner diameter × 150 mm C18 reversed-phase column (Nikkyo Technos) using a segmented linear gradient: 5%–20% ACN from 0 to 40 min, 20%–35% ACN from 40 to 60 min, followed by a rapid increase to 95% ACN from 60 to 61 min, and held at 95% ACN from 61 to 75 min. Data acquisition was conducted in dia-PASEF mode with an MS1 m/z range of 100–1700 and an ion mobility (1/K0) range of 0.6–1.6. The ramp time was set to 100 ms with a 100% duty cycle. The MS2 polygon was manually defined based on the region where peptides were predominantly detected, specifically using the following four vertices: Point 1 (m/z = 300, 1/K0 = 0.64), Point 2 (m/z = 300, 1/K0 = 0.8), Point 3 (m/z = 770, 1/K0 = 0.97), and Point 4 (m/z = 770, 1/K0 = 1.15). Within the defined polygon, an 8 Da isolation window was employed with an overlap of 0.5 Da for each window, resulting in 62 windows per cycle and a cycle time of 3.36 s. Protein identification was carried out in DIA-NN (version 1.9)81 against a Drosophila melanogaster (UP000000803) in silico spectral library. Parameters for constructing the library included: trypsin as the digestion enzyme, one allowed missed cleavage, peptide lengths ranging from 7 to 45 amino acids, precursor charges of 2 to 4, and a fragment ion m/z range of 200–1800. Features such as library-free search, deep learning-based retention time and ion mobility predictions, N-terminal methionine cleavage, and cysteine carbamidomethylation were enabled. In the DIA-NN search settings, MS1 and MS2 mass accuracies were set to auto, neural network classifiers were configured for single-pass mode, and the quantification strategy was set to QuantUMS (high precision).

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

SUBMITTER: Mikiko C. Siomi

PROVIDER: PXD074025 | JPOST Repository | Wed Feb 04 00:00:00 GMT 2026

REPOSITORIES: jPOST

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	PJ1691_Hirakata_1_KD_E1_IPMS_timsHT_DIA_Slot2-10_1_5416.d.zip	Other
	PJ1691_Hirakata_2_KD_E2_IPMS_timsHT_DIA_Slot2-12_1_5420.d.zip	Other
	PJ1691_Hirakata_3_KD_E3_IPMS_timsHT_DIA_Slot2-14_1_5424.d.zip	Other
	PJ1691_Hirakata_4_KD_Sh1_IPMS_timsHT_DIA_Slot2-11_1_5418.d.zip	Other
	PJ1691_Hirakata_5_KD_Sh2_IPMS_timsHT_DIA_Slot2-13_1_5422.d.zip	Other

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Publications

Transient residence of the repulsive client Shutdown in Yb bodies plays a critical role in Piwi-piRISC biogenesis and maintaining fertility.

Hirakata Shigeki S Fukaya Takashi T Fujita Aoi A Kosako Hidetaka H Siomi Mikiko C MC

Molecular cell 20260401 7

The formation of biomolecular condensates, such as Yb bodies in Drosophila ovarian somatic cells, is significantly contributed to by phase separation of scaffold proteins. Client proteins transiently accumulate via interactions with these scaffolds; however, how client flux is regulated remains unclear. Here, we investigate Shutdown, a client protein of Yb bodies-the site of Piwi-piRNA-induced silencing complex (piRISC) precursor (pre-Piwi-piRISC) formation-and show that cytosolic Shutdown conne ...[more]

PMID: 41932311

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