PXD041241

PXD041241 is an original dataset announced via ProteomeXchange.

Title	LC-MS/MS analysis of co-immunoprecipitates with PINK1-3FLAG
Description	Parental HeLa cells and HeLa cells stably expressing PINK1-3FLAG were treated with or without 10 μM CCCP for 3 h, incubated with 0.1% paraformaldehyde for 10 min at room temperature, and then quenched with 100 mM glycine-NaOH (pH7.5) for 4 min. After washing twice with 20 mM HEPES-NaOH (pH7.4) and 137 mM NaCl, the cells were solubilized on ice for 10 min in RIPA buffer (20 mM HEPES-NaOH [pH 7.5], 150 mM NaCl, 1mM MgCl2, 1 mM EGTA, 0.05% SDS, 0.25% sodium deoxycholate, 1% NP-40) supplemented with a protease inhibitor cocktail and 50 unit/ml Benzonase (Merck Millipore), and then subjected to centrifugation (20,000 x g) at 4°C for 15 min. The resultant supernatants were incubated at 4°C for 1 h with anti-FLAG (M2) mAb magnetic beads (Sigma-Aldrich). The beads were washed four times with 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 digests were reduced, alkylated, acidified, and desalted with GL-Tip SDB (GL Sciences). The eluates were evaporated and dissolved in 0.1% trifluoroacetic acid and 3% acetonitrile. LC-MS/MS analysis of the resultant peptides was performed on an EASY-nLC 1200 UHPLC connected to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source (Thermo Fisher). The peptides were separated on a 75-μm inner diameter x 150 mm C18 reversed-phase column (Nikkyo Technos) with a linear 4%-32% acetonitrile gradient for 0-150 min followed by an increase to 80% acetonitrile for 15 min and finally held at 80% acetonitrile for 15 min. The mass spectrometer was operated in data-dependent acquisition mode with the top 10 MS/MS method. MS1 spectra were measured at a resolution of 70,000, an automatic gain control target of 1e6, and a mass range from 350 to 1500 m/z. HCD MS/MS spectra were triggered at a resolution of 17,500, an automatic gain control target of 5e4, an isolation window of 2.0 m/z, a maximum injection time of 60 ms, and a normalized collision energy of 27. Dynamic exclusion was set to 10 s. Raw data were directly analyzed against the SwissProt database restricted to Homo sapiens using Proteome Discoverer 2.4 (Thermo Fisher) with the 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 0.02 Da; (d) carbamidomethylation of cysteine as a fixed modification; and (e) acetylation of 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 based on 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	2023-04-07
AnnouncementXML	Submission_2023-08-21_23:52:06.616.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	instrument

Revision	Datetime	Status	ChangeLog Entry
0	2023-03-31 01:37:11	ID requested
1	2023-04-06 15:03:30	announced
⏵ 2	2023-08-21 23:52:07	announced	2023-08-22: Updated PubMed.

Akabane S, Watanabe K, Kosako H, Yamashita SI, Nishino K, Kato M, Sekine S, Kanki T, Matsuda N, Endo T, Oka T, TIM23 facilitates PINK1 activation by safeguarding against OMA1-mediated degradation in damaged mitochondria. Cell Rep, 42(5):112454(2023) [pubmed]

submitter keyword: IP-MS, anti-FLAG antibody, PINK1

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

jPOST dataset URI

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