PXD039312

PXD039312 is an original dataset announced via ProteomeXchange.

Title	TMTpro-18plex analysis of siSTK38 or siLuc HeLa cells treated with EBSS or LLOMe
Description	HeLa cells were transfected with STK38 siRNA or Luc siRNA and then treated with EBSS or LLOMe in three biological replicates. The cells were lysed in 500 µL guanidine buffer (6 M guanidine-HCl, 100 mM HEPES-NaOH, pH 7.5, 10 mM TCEP, 40 mM chloroacetamide). After heating and sonication, proteins (330 µg each) were purified by methanol–chloroform precipitation and resuspended in 37 µL 0.1% RapiGest SF (Waters) in 50 mM triethylammonium bicarbonate. After sonication and heating at 95 ºC for 10 min, the proteins were digested with 5 µg trypsin/Lys-C mix (Promega) at 37 ºC overnight. The digested peptides (200 µg each) were labeled with 0.5 mg TMTpro-18plex reagents (Thermo Fisher Scientific) for 1 h at 25 ºC. After the reaction was quenched with hydroxylamine, all the TMT-labeled samples were pooled, acidified with TFA, and subjected to High-Select Fe-NTA phosphopeptide enrichment kit (Thermo Fisher Scientific). The eluate was fractionated by offline high-pH reversed-phase chromatography on a Vanquish DUO UHPLC (Thermo Fisher Scientific). Briefly, the peptides were loaded onto a 4.6 × 250 mm Xbridge BEH130 C18 column with 3.5 mm particles (Waters) and separated using a 30 min multistep gradient of solvents A (10 mM ammonium formate at pH 9.0 in 2% ACN) and B (10 mM ammonium formate pH 9.0 in 80% ACN), at a flow rate of 1 mL/min. Peptides were separated into 48 fractions, which were consolidated into 16 fractions. Each fraction was evaporated in a SpeedVac concentrator and dissolved 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 a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on the analytical column (75 μm × 15 cm, 3 μm; Nikkyo Technos) with a linear gradient of 4–32% for 0–100 min, followed by an increase to 80% ACN for 10 min and finally held at 80% ACN for 10 min. The mass spectrometer was operated in data-dependent acquisition mode with a top 10 MS/MS method. MS1 spectra were measured with a resolution of 70,000, an AGC target of 3e6, and a mass range from 375 to 1,400 m/z. MS/MS spectra were triggered at a resolution of 35,000, an AGC target of 1e5, an isolation window of 0.7 m/z, a maximum injection time of 150 ms, and a normalized collision energy of 33. Dynamic exclusion was set to 20 s. Raw data were directly analyzed against the SwissProt database restricted to Homo sapiens using Proteome Discoverer version 2.5 with the Sequest HT search engine for identification and TMT quantification. 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) TMTpro of lysine and peptide N-terminus and carbamidomethylation of cysteine as fixed modifications; (e) oxidation of methionine and phosphorylation of serine, threonine, and tyrosine as variable modifications. Peptides were filtered at an FDR of 1% using the Percolator node. TMT quantification was performed using the Reporter Ions Quantifier node. Normalization was performed such that the total sum of the abundance values for each TMT channel over all peptides was the same.
HostingRepository	jPOST
AnnounceDate	2023-11-21
AnnouncementXML	Submission_2023-12-05_05:09:10.011.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; unknown modification; unknown modification; O-phospho-L-serine; O-phospho-L-threonine; O4'-phospho-L-tyrosine
Instrument	Q Exactive Plus

Revision	Datetime	Status	ChangeLog Entry
0	2023-01-06 22:38:51	ID requested
1	2023-11-21 04:12:30	announced
⏵ 2	2023-12-05 05:09:10	announced	2023-12-05: Updated PubMed.

Ogura M, Kaminishi T, Shima T, Torigata M, Bekku N, Tabata K, Minami S, Nishino K, Nezu A, Hamasaki M, Kosako H, Yoshimori T, Nakamura S, Microautophagy regulated by STK38 and GABARAPs is essential to repair lysosomes and prevent aging. EMBO Rep, 24(12):e57300(2023) [pubmed]

submitter keyword: TMTpro-18plex, STK38 siRNA, EBSS, LLOMe

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

jPOST dataset URI

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