PXD063706

PXD063706 is an original dataset announced via ProteomeXchange.

Title	LFQ of carboxymethyl/carboxyethyl lysine peptides from MGO- or GO-treated HeLa cells
Description	HeLa cells (n=2) were treated with cell culture media containing 2 mM MGO or GO for 2 h and lysed in 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 (300 µg 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 3 μg trypsin/Lys-C mix (Promega) at 37 ºC. The resulting peptide solutions were diluted 6-fold with HBS (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl), centrifuged, and used with a PTMScan Carboxymethyl/Carboxyethyl Lysine Motif kit (Cell Signaling Technology). The eluates in 0.15% TFA and 5% acetonitrile were desalted, evaporated, and re-dissolved 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 5% B and ramping linearly to 20% B by 40 min, then to 35% B by 60 min, followed by a rapid increase to 95% B at 61 min, where it was held until 65 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); and carboxymethylation (+58.0055 Da) or carboxyethylation (+72.0211 Da) of 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_2025-05-06_23:10:44.257.xml
DigitalObjectIdentifier
ReviewLevel	Non 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; N6-carboxymethyl-L-lysine; N6-1-carboxyethyl-L-lysine
Instrument	instrument

Revision	Datetime	Status	ChangeLog Entry
0	2025-05-06 23:09:26	ID requested
⏵ 1	2025-05-06 23:10:44	announced

Dataset with its publication pending

submitter keyword: carboxymethylation, carboxyethylation, MGO, GO

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

jPOST dataset URI

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