PXD063699

PXD063699 is an original dataset announced via ProteomeXchange.

Title	LFQ of 3-phosphoglyceroyl lysine peptides from WT and DJ-1 KO HeLa cells
Description	WT or DJ-1 KO HeLa cells (n=3) were lysed in 500 μL of 6 M guanidine-HCl, 100 mM HEPES-NaOH, pH 7.5, 10 mM TCEP, and 40 mM CAA. The lysates were dissolved by heating and sonication, followed by centrifugation at 20,000 x g for 15 min at 4 ºC. The supernatants were recovered and proteins (1 mg each) purified by methanol–chloroform precipitation were solubilized in 150 μL of 0.1% RapiGest (Waters) in 50 mM triethylammonium bicarbonate. After sonication, the protein solutions were digested overnight with 10 μg trypsin/Lys-C mix (Promega) at 37 ºC. The resulting peptide solutions were acidified with TFA, centrifuged, and used with a High-Select Fe-NTA phosphopeptide enrichment kit (Thermo Fisher Scientific). The eluates were acidified, desalted using GL-Tip SDB (GL Sciences), evaporated in a SpeedVac concentrator, and redissolved in 0.1% TFA and 3% acetonitrile. LC-MS/MS analysis of the resultant peptides was performed on a nanoElute 2 coupled with a timsTOF HT mass spectrometer (Bruker). The peptides were separated on a 75-μm inner diameter x 150 mm C18 reversed-phase column (Nikkyo Technos). The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Peptides were loaded onto the column at a flow rate of 0.2 μL/min starting at 3% B, which was linearly ramped to 32% B over 90 min, then raised to 95% B at 91 min, and held at that level until 101 min. The mass spectrometer was operated in parallel accumulation–serial fragmentation (PASEF) mode. The m/z range for both MS1 and MS2 spectra was 100-1700, and the ion mobility range was 0.6-1.6 V.s/cm3. The ramp time was 100 ms, with a duty cycle of 100%. Each acquisition cycle consisted of 10 PASEF MS2 scans. A polygon filter was applied to the m/z and ion mobility space to exclude low m/z, singly charged ions from precursor selection. The raw data were processed using FragPipe (v22.0). Database searches were performed with MSFragger (v4.1), employing the default parameters of the LFQ-phospho workflow against the UniProt human database (20,454 entries). Carbamidomethylation of cysteine (+57.0215 Da) was set as a fixed modification. The following variable modifications were included: acetylation of the protein N-terminus (+42.0106 Da); oxidation of methionine (+15.9949 Da); phosphorylation (+79.9663 Da) of serine, threonine, or tyrosine; and 3-phosphoglyceroyl (+167.9824 Da) at lysine. The resulting identifications were filtered using Philosopher with default parameters (MS Booster was disabled), and IonQuant (v1.10.27) was used for quantification with default software settings.
HostingRepository	jPOST
AnnounceDate	2025-05-07
AnnouncementXML	Submission_2025-06-06_19:41:52.055.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; L-methionine sulfoxide; O-phospho-L-serine; O-phospho-L-threonine; O4'-phospho-L-tyrosine; unknown modification
Instrument	instrument

Revision	Datetime	Status	ChangeLog Entry
0	2025-05-06 22:30:49	ID requested
1	2025-05-06 23:10:12	announced
⏵ 2	2025-06-06 19:41:54	announced	2025-06-07: Updated PubMed.

Watanabe A, Ogiwara S, Saito M, Mishima M, Yamashina M, Ishitani R, Ito Y, Tanaka K, Koyano F, Yamano K, Kosako H, Moriwaki Y, Matsuda N, The reaction mechanism for glycolysis side product degradation by Parkinson's disease-linked DJ-1. J Cell Biol, 224(8):(2025) [pubmed]

submitter keyword: 3-phosphoglyceroylation, DJ-1

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

jPOST dataset URI

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