Dataset Information

LFQ of immunoprecipitates with GFP-Trap from GFP-, OfMasc-GFP-, or Del1-OfMasc-GFP-transfected cells

ABSTRACT: OfMasc-GFP derivatives or GFP were transiently expressed in OfTC cells. At 2 days post transfection, OfTC cells were treated with 100 nM Bortezomib for 1 day. The OfT1C cells were then fixed with 0.1% formaldehyde, and the fixation was quenched with 300 mM glycine. The cells were lysed on ice in 1 mL of RIPA buffer. After centrifugation, the supernatants were incubated with GFP-Trap Magnetic Agarose (ChromoTek) for 3 h at 4ºC. Proteins bound to the beads were digested by adding 200 ng of Trypsin/Lys-C mix (Promega) for 16 h at 37ºC. The digests were reduced, alkylated, acidified with trifluoroacetic acid, and desalted using a GL-Tip SDB (GL Sciences). LC-MS/MS analysis of the resultant peptides was performed using an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer equipped with a nanoelectrospray ion source (Thermo Fisher Scientific). Raw data were directly analyzed against the wFur encoded protein data predicted from an in-house assembled wFur draft genomic sequence and O. furnacalis protein data ‘GCF_004193835.1_ASM419383v1_protein.faa’ downloaded from NCBI supplemented with OfMasc-GFP and GFP-Trap sequences using Proteome Discoverer version 2.4 (Thermo Fisher Scientific) with Sequest HT search engine. Label-free precursor ion quantification was performed using the precursor ions quantifier node.

ORGANISM(S): Cellular Organisms

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD034837 | JPOST Repository | Tue Oct 04 00:00:00 BST 2022

REPOSITORIES: jPOST

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	107_Matsuda_1_GFP_Trap_GFP_FusionIT.raw	Raw
	109_Matsuda_2_GFP_Trap_OfMasc_GFP_FusionIT.raw	Raw
	111_Matsuda_3_GFP_Trap_Del1_FusionIT.raw	Raw
	210415_Matsuda_OfMasc_GFP_FusionIT-(1).pdResult	Other

Items per page:

1 - 4 of 4

Publications

A Wolbachia factor for male killing in lepidopteran insects.

Katsuma Susumu S Hirota Kanako K Matsuda-Imai Noriko N Fukui Takahiro T Muro Tomohiro T Nishino Kohei K Kosako Hidetaka H Shoji Keisuke K Takanashi Hideki H Fujii Takeshi T Arimura Shin-Ichi SI Kiuchi Takashi T

Nature communications 20221114 1

Bacterial symbionts, such as Wolbachia species, can manipulate the sexual development and reproduction of their insect hosts. For example, Wolbachia infection induces male-specific death in the Asian corn borer Ostrinia furnacalis by targeting the host factor Masculinizer (Masc), an essential protein for masculinization and dosage compensation in lepidopteran insects. Here we identify a Wolbachia protein, designated Oscar, which interacts with Masc via its ankyrin repeats. Embryonic expression o ...[more]

PMID: 36376299

Similar Datasets

Project description: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.