PXD072519-1

PXD072519 is an original dataset announced via ProteomeXchange.

Title	DIA-PASEF–based FLAG-IP proteomic analysis of nuclear or cytosolic p62 condensates in HeLa cells
Description	Cells were cross-linked with 0.1% formaldehyde for 10 min at 25 °C and quenched with 100 mM glycine. After washing with HBS (20 mM HEPES-NaOH, pH 7.5, 150 mM NaCl), cells were lysed in HEPES-RIPA buffer (20 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 1 mM MgCl₂, 1 mM EGTA, 1% NP-40, 0.25% sodium deoxycholate, 0.05% SDS) supplemented with EDTA-free protease inhibitors, phosphatase inhibitors, and Benzonase. Lysates were clarified by centrifugation (4 ºC), and supernatants were incubated with anti-FLAG M2 magnetic beads (Sigma, M8823) for 2 h at 4 °C with rotation. Beads were washed three times with HEPES-RIPA buffer and twice with 50 mM ammonium bicarbonate. On-bead digestion was performed with 200 ng of trypsin/Lys-C mix overnight at 37 °C. Peptides were reduced, alkylated, acidified, desalted using GL-Tip SDB, dried, and reconstituted in 0.1% TFA and 3% 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) with a linear 5%–35% ACN gradient for 0–60 min followed by an increase to 95% ACN for 1 min and a final hold at 80% ACN for 4 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) against a human 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).
HostingRepository	jPOST
AnnounceDate	2025-12-30
AnnouncementXML	Submission_2025-12-30_06:07:01.302.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Hidetaka Kosako
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	L-methionine sulfoxide; Acetyl; S-carboxamidomethyl-L-cysteine
Instrument	instrument

Revision	Datetime	Status	ChangeLog Entry
0	2025-12-30 06:06:41	ID requested
⏵ 1	2025-12-30 06:07:01	announced
2	2026-02-07 03:54:15	announced	2026-02-07: Updated PubMed.

Dataset with its publication pending

submitter keyword: DIA-PASEF, FLAG-IP, p62 condensates, HeLa cells

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

jPOST dataset URI

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