PXD056735

PXD056735 is an original dataset announced via ProteomeXchange.

Title	IP-MS of ASXL1 without fractionation using 293T cells and THP-1 cells
Description	The cell lysates were mixed with anti-FLAG-antibody-crosslinked Dynabeads. The beads-lysate mix was incubated at 4°C for 45 min. After washing with PBS-T, proteins were eluted with Laemmli’s SDS ample buffer by incubating at 37°C for 30 min. The collected supernatants were frozen at -80°C as samples for LC-MS. After reduction with 10 mM TCEP at 100°C for 10 min and alkylation with 50 mM iodoacetamide at ambient temperature for 45 min, the protein samples were subjected to digestion with Trypsin/Lys-C Mix (Promega) at 47°C for 2 h on S-Trap columns (ProtiFi, NY, USA). The resulting peptides were extracted from gel fragments and analysed using an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) combined with UltiMate 3000 RSLC nano-flow HPLC (Thermo Scientific). The peptides were enriched with μ-Precolumn (0.3 mm i.d. × 5 mm, 5 μm, Thermo Scientific) and separated on an AURORA column (0.075 mm i.d. × 250 mm, 1.6 μm, Ion Opticks Pty Ltd, Australia) using the two-step gradient 2-40% acetonitrile for 110 min, followed by 40-95% acetonitrile for 5 min in the presence of 0.1% formic acid. The compensation voltages for the gas-phase fractionation by FAIMS Pro (Thermo Scientific) were set at -40, -60 and -80 V. The analytical parameters of the Orbitrap Fusion Lumos were set as follows: resolution of full scans = 50,000, scan range (m/z) = 350-1500, maximum injection time of full scans = 50 ms, AGC target of full scans = 4 × 10^5, dynamic exclusion duration = 30 s, cycle time of data dependent MS/MS acquisition = 2 s, activation type = HCD, detector of MS/MS = ion trap, maximum injection time of MS/MS = 35 ms, and AGC target of MS/MS = 1 × 10^4. The MS/MS spectra were searched against the Homo sapiens protein sequence database in SwissProt using Proteome Discoverer 3.0 software (Thermo Scientific), in which peptide identification filters were set at “false discovery rate < 1%”. Label-free relative quantification analysis for proteins was performed with the default parameters of Minora Feature Detector node, Feature Mapper node, and Precursor Ions Quantifier node in Proteome Discoverer 3.0 software.
HostingRepository	jPOST
AnnounceDate	2024-10-30
AnnouncementXML	Submission_2024-10-29_11:14:49.388.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Naru Sato
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	S-carboxamidomethyl-L-cysteine; L-methionine sulfoxide
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2024-10-11 09:31:45	ID requested
⏵ 1	2024-10-29 11:14:49	announced

Dataset with its publication pending

submitter keyword: ASXL1, Clonal hematopoiesis, Atherosclerosis, Monocyte, Macrophage, Inflammatory signalling, PPI

Toshio Kitamura
lab head
Naru Sato
contact affiliation	University of Tokyo
dataset submitter

jPOST dataset URI

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