PXD074028-1

PXD074028 is an original dataset announced via ProteomeXchange.

Title	FLAG IP-MS analysis of MORC1-interacting proteins in NIH3T3 cells
Description	NIH3T3 cells expressing 3xFlag-mCherry or 3xFlag-MORC1 WT (n = 3 each) were harvested by centrifugation and resuspended in hypotonic buffer [20 mM HEPES-KOH (pH 7.3), 10 mM KCl, 1.5 mM MgCl₂, 0.5 mM DTT, 2 µg/mL pepstatin A, 2 µg/mL leupeptin, and 0.5 µg/mL aprotinin]. The suspension was pipetted and passed through a 25-gauge needle three times, then centrifuged, and the pellet was washed three times with hypotonic buffer. The pellet was resuspended in co-IP buffer [50 mM HEPES-KOH (pH 7.3), 200 mM KCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 2 µg/mL pepstatin A, 2 µg/mL leupeptin, and 0.5 µg/mL aprotinin] and sonicated (Branson digital sonifier 250D-Advanced; amplitude 20%, 0.2 s ON / 0.8 s OFF cycles, total ON-time 1 min). Lysates were clarified by centrifugation at ~20,000 × g for 20 min at 4°C. Dynabeads Protein G (Thermo Fisher Scientific) were washed with co-IP buffer and incubated with Flag M2 antibody (Sigma-Aldrich) for ≥30 min at room temperature with rotation. The clarified lysate was incubated with the antibody-bound beads for 2 h at 4°C with rotation. Beads were washed five times with co-IP buffer. Proteins bound to the beads were digested with 200 ng of trypsin/Lys-C mix (Promega) at 37°C overnight. The resulting peptides were reduced, alkylated, acidified, and desalted using GL-Tip SDB (GL Sciences). Eluates were dried in a SpeedVac concentrator and reconstituted in 0.1% trifluoroacetic acid and 3% acetonitrile (ACN). LC-MS/MS analysis was performed on an EASY-nLC 1200 UHPLC system coupled to an Orbitrap Fusion mass spectrometer equipped with a nanoelectrospray ion source (Thermo Fisher Scientific). Peptides were separated on a 75 µm inner diameter × 150 mm C18 reversed-phase column (Nikkyo Technos) with a linear 4–32% ACN gradient for 0–100 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 with a maximum duty cycle of 3 s. MS1 spectra were acquired at a resolution of 120,000 with an automatic gain control (AGC) target of 4e5 and a mass range of 375–1,500 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 35 ms, and a normalized collision energy of 30. Dynamic exclusion was set to 20 s. Raw data were searched against the SwissProt database restricted to Mus musculus using Proteome Discoverer version 2.5 (Thermo Fisher Scientific) with the Sequest HT search engine. Search parameters were as follows: trypsin as the enzyme with up to two missed cleavages, precursor mass tolerance of 10 ppm, fragment mass tolerance of 0.6 Da, carbamidomethylation of cysteine as a fixed modification, and acetylation of 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 performed using the Precursor Ions Quantifier node, and normalization was applied so that the total sum of abundance values for each sample over all peptides was the same.
HostingRepository	jPOST
AnnounceDate	2026-08-12
AnnouncementXML	Submission_2026-08-11_21:59:53.699.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Hidetaka Kosako
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	L-methionine sulfoxide; Acetyl; S-carboxamidomethyl-L-cysteine
Instrument	Orbitrap Fusion

Revision	Datetime	Status	ChangeLog Entry
0	2026-02-03 22:31:52	ID requested
⏵ 1	2026-08-11 21:59:54	announced

Dataset with its publication pending

submitter keyword: FLAG, IP-MS, MORC1, NIH3T3

Mikiko C. Siomi
lab head
Hidetaka Kosako
contact affiliation	Tokushima University
dataset submitter

jPOST dataset URI

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.jpostdb.org/JPST004383/