PXD052134

PXD052134 is an original dataset announced via ProteomeXchange.

Title	anti-HA IP-MS of Pdzd8-3×HA knock-in mouse neocortex
Description	Pdzd8-3xHA knock-in mice and control littermates at postnatal 10 days were put to sleep using medetomidine hydrochloride, midazolam and butorphanol. Pups were then put on the ice for 5 minutes and exsanguinated by terminal intracardial perfusion with ice-cold 2% paraformaldehyde in PBS. The neocortex was then removed and sonicated five times for 30 seconds with ice-cold lysis buffer (20 mM Hepes-NaOH pH7.5, 1 mM EGTA, 1 mM MgCl2, 150 mM NaCl, 0.25% Na-deoxycholate, 0.05% SDS, 1% NP40, Benzonase, PhosSTOP phosphatase inhibitor and cOmplete protease inhibitor cocktail). After the lysates were centrifuged at 20,000 g for 15 min at 4ºC, the resulting supernatants were incubated for 3 hours at 4ºC with a 2.5 μL slurry of Sera-Mag SpeedBeads Protein A/G pre-incubated with 2.5 μL of anti-HA-tag rabbit mAb (Cell signaling technology, C29F4). The beads were washed four times with the lysis buffer and then twice with 50 mM ammonium bicarbonate. Proteins on the beads were digested by adding 200 ng trypsin/Lys-C mix (Promega) at 37ºC overnight. The resulting digests were reduced, alkylated, acidified, and desalted using GL-Tip SDB (GL Sciences). The eluates were evaporated and dissolved in 0.1% trifluoroactic acid and 3% ACN. LC-MS/MS analysis of the resultant peptides was performed on an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on a C18 reversed-phase column (75 mm [inner diameter] x 150 mm; Nikkyo Technos) with a linear 4%–32% ACN gradient for 0–100 minutes, followed by an increase to 80% ACN for 10 minutes and final hold at 80% ACN for 10 minutes. The mass spectrometer was operated in 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 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 directly analyzed against the SwissProt database restricted to Mus musculus using Proteome Discoverer version 2.5 (Thermo Fisher Scientific) with Sequest HT search engine for identification and label-free precursor ion quantification. The search parameters were as follows: (i) trypsin as an enzyme with up to two missed cleavages; (ii) precursor mass tolerance of 10 ppm; (iii) fragment mass tolerance of 0.6 Da; (iv) carbamidomethylation of cysteine as a fixed modification; and (v) acetylation of the protein N-terminus and oxidation of methionine as variable modifications. Peptides and proteins were filtered at a false discovery rate (FDR) of 1% using the Percolator node and Protein FDR Validator node, respectively. Label-free quantification was performed on the basis of the intensities of precursor ions using the Precursor Ions Quantifier node. Normalization was performed such that the total sum of abundance values for each sample over all peptides was the same.
HostingRepository	jPOST
AnnounceDate	2025-04-18
AnnouncementXML	Submission_2025-04-17_22:51:32.837.xml
DigitalObjectIdentifier
ReviewLevel	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	2024-05-09 00:28:47	ID requested
1	2025-04-17 19:39:33	announced
⏵ 2	2025-04-17 22:51:33	announced	2025-04-18: Updated PubMed.

submitter keyword: HA, IP-MS, Pdzd8, neocortex

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

jPOST dataset URI

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