PXD040970

PXD040970 is an original dataset announced via ProteomeXchange.

Title	LFQ of mNeonGreen-Trap IP-MS from cells expressing HKDC1-mNeonGreen
Description	Cells stably expressing full-length HKDC1-mNeonGreen or △N20 HKDC1-mNeonGreen proteins were grown in a 10-cm dish and were treated with or without antimycin/oligomycin for 3h, and then cross-linked with 0.1% formaldehyde for 10min at room temperature. After cross-linking was quenched with 100mM glycine for 10min at room temperature, cells were washed with HEPES-saline containing 20mM HEPES-NaOH (pH7.5) and 137mM NaCl, and lysed on ice in HEPES-RIPA buffer containing 20mM HEPES-NaOH (pH7.5), 150mM NaCl, 1mM EGTA, 1mM MgCl2, 0.25% (w/v) Na-deoxycholate, 0.05% SDS, 1% (v/v) NP-40, 0.2% (v/v) Benzonase Nuclease (70746, Millipore), PhosSTOP (4906837001, Roche) and Protease Inhibitor Cocktail. After sonication and centrifugation at 15,000rpm, 4°C for 15min, the supernatants were incubated with mNeonGreen-Trap magnetic agarose beads (ntma-20, ChromoTek) for 2 h at 4°C. The beads were washed four times with HEPES-RIPA 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 37°C overnight. The resulting digests were reduced, alkylated, acidified, and desalted using GL-Tip SDB. The eluates were evaporated and dissolved in 0.1% trifluoroacetic acid and 3% acetonitrile (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 µm x 150 mm; Nikkyo Technos) with a linear 4-32% ACN gradient for 0-100 min, followed by an increase to 80% ACN for 10 min and 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 measured with a resolution of 120,000, an 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 Homo sapiens using Proteome Discoverer 2.5 (Thermo Fisher Scientific) with Sequest HT search engine. The search parameters were as follows: (a) trypsin as an enzyme with up to two missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.6 Da; (d) carbamidomethylation of cysteine as a fixed modification; (e) 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 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	2024-01-21
AnnouncementXML	Submission_2024-01-20_17:09:25.828.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Hidetaka Kosako
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	S-carboxamidomethyl-L-cysteine; L-methionine sulfoxide; Acetyl
Instrument	Orbitrap Fusion

Revision	Datetime	Status	ChangeLog Entry
0	2023-03-20 00:37:33	ID requested
⏵ 1	2024-01-20 17:09:26	announced

Cui M, Yamano K, Yamamoto K, Yamamoto-Imoto H, Minami S, Yamamoto T, Matsui S, Kaminishi T, Shima T, Ogura M, Tsuchiya M, Nishino K, Layden BT, Kato H, Ogawa H, Oki S, Okada Y, Isaka Y, Kosako H, Matsuda N, Yoshimori T, Nakamura S, HKDC1, a target of TFEB, is essential to maintain both mitochondrial and lysosomal homeostasis, preventing cellular senescence. Proc Natl Acad Sci U S A, 121(2):e2306454120(2024) [pubmed]

submitter keyword: LFQ, mNeonGreen-Trap, HKDC1

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

jPOST dataset URI

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