PXD052135

PXD052135 is an original dataset announced via ProteomeXchange.

Title	Proximity biotinylation of PDZD8-TurboID KI HeLa cells
Description	PDZD8-TurboID knocked-in HeLa cells were plated into a 15 cm dish and cultured two overnight. The cells were treated with Biotin at the final concentration of 50 μM, and incubated for 6 hours. The cells were washed twice with ice-cold Hepes-saline and lysed in 6 M guanidine-HCl containing 100 mM HEPES-NaOH (pH7.5), 10 mM TCEP, and 40 mM chloroacetamide. After heating and sonication, proteins (1.3 mg each) were purified by methanol–chloroform precipitation and resuspended in 200 μL of PTS buffer (12 mM SDC, 12 mM SLS, 100 mM Tris-HCl, pH8.0). After sonication and heating at 95ºC for 10 minutes, the protein solutions were diluted 5-fold with 100 mM Tris-HCl, pH8.0 and digested with 13 μg of trypsin (Pierce) at 37ºC overnight. After heating at 95ºC for 10 minutes, the digested peptides were incubated with the ACN-prewashed Tamavidin 2-REV beads (FUJIFILM Wako) for 3 hours at 4ºC. After washing five times with TBS (50 mM Tris-HCl, pH7.5, 150 mM NaCl), biotinylated peptides were eluted for 15 minutes at 37ºC twice with 100 μL of 1 mM biotin in TBS. The combined eluates were desalted using GL-Tip SDB, evaporated, and redissolved in 0.1% TFA 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. The peptides were separated on the C18 reversed-phase column with a linear 4%–32% ACN gradient for 0–60 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 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 200 ms, and a normalized collision energy of 30. Dynamic exclusion was set to 10 s. Raw data were directly analyzed against the SwissProt database restricted to Mus musculus using Proteome Discoverer version 2.5 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, oxidation of methionine, and biotinylation of lysine as variable modifications. Peptides and proteins were filtered at a 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_22:51:42.838.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; Acetyl; N6-biotinyl-L-lysine; L-methionine sulfoxide
Instrument	Orbitrap Fusion

Revision	Datetime	Status	ChangeLog Entry
0	2024-05-09 00:46:44	ID requested
1	2025-04-17 19:39:16	announced
⏵ 2	2025-04-17 22:51:43	announced	2025-04-18: Updated PubMed.

Nakamura K, Aoyama-Ishiwatari S, Nagao T, Paaran M, Obara CJ, Sakurai-Saito Y, Johnston J, Du Y, Suga S, Tsuboi M, Nakakido M, Tsumoto K, Kishi Y, Gotoh Y, Kwak C, Rhee HW, Seo JK, Kosako H, Potter C, Carragher B, Lippincott-Schwartz J, Polleux F, Hirabayashi Y, Mitochondrial complexity is regulated at ER-mitochondria contact sites via PDZD8-FKBP8 tethering. Nat Commun, 16(1):3401(2025) [pubmed]

submitter keyword: TurboID, PDZD8, HeLa

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

jPOST dataset URI

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