PXD052654-1

PXD052654 is an original dataset announced via ProteomeXchange.

Title	TMT-based phosphoproteomics of mouse hippocampus
Description	Brain tissues were homogenized in 6 M guanidine-HCl containing 100 mM HEPES−NaOH (pH 7.5), 10 mM Tris (2-carboxyethyl) phosphine (TCEP), and 40 mM chloroacetamide. After heating and sonication, proteins were purified by methanol−chloroform precipitation and solubilized with 0.1% 21 RapiGest SF in 50 mM triethylammonium bicarbonate. The proteins were digested with trypsin/Lys-C mix (Promega) at 37 ºC overnight. 250 µg of digested peptides from each sample were labeled with TMT 10plex reagents (Thermo Fisher Scientific). Labeled peptides were enriched using a High-Select Fe-NTA Phosphopeptide Enrichment Kit (Thermo Fisher Scientific). The labeled peptides were pooled and fractionated using a Pierce High pH reversed-phase peptide fractionation kit (Thermo Fisher Scientific). The labeled peptides were pooled and fractionated using offline high-pH reversed-phase chromatography on a Vanquish DUO UHPLC system (Thermo Fisher Scientific). Chromatographic separation was performed on a C18 reversed-phase column (4.6 × 250 mm Xbridge BEH130 C18 column with 3.5 mm particles; Waters) with a multistep gradient of mobile phase A (10 mM ammonium formate at pH 9.0 in 2% ACN) and B (10 mM ammonium formate pH 9.0 in 80% ACN). 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. LC-MS/MS analysis 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 a C18 reversed-phase column (75 μm × 150 mm; Nikkyo Technos) with a linear gradient of 4−20% for 0−115 min and 20−32% for 115−160 min, followed by an increase to 80% ACN for 10 min and finally a hold at 80% ACN for 10 min. The mass spectrometer was operated in datadependent 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 1400 m/z. MS/MS spectra were acquired 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 34. Dynamic exclusion was set to 20 s. Raw MS data were searched against the SwissProt database restricted to Mus musculus using Proteome Discoverer version 2.4 with the Sequest HT search engine. Analysis of MS spectra was performed using the following parameters: (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) TMT of lysine and peptide N-terminus and carbamidomethylation of cysteine as fixed modifications; and (e) phosphorylation of serine, threonine and tyrosine and oxidation of methionine as variable modifications. Peptides were filtered at a false discovery rate of 1% using the Percolator node.
HostingRepository	jPOST
AnnounceDate	2024-08-20
AnnouncementXML	Submission_2024-08-20_02:44:24.002.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Masanori Tachikawa
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	S-carboxamidomethyl-L-cysteine; L-methionine sulfoxide; O-phospho-L-serine; O-phospho-L-threonine; O4'-phospho-L-tyrosine; TMT6plex reporter+balance reagent N-acylated residue; TMT6plex reporter+balance reagent acylated N-terminal
Instrument	Q Exactive Plus

Revision	Datetime	Status	ChangeLog Entry
0	2024-05-28 23:43:53	ID requested
⏵ 1	2024-08-20 02:44:24	announced

Dataset with its publication pending

submitter keyword: TMT, phosphoproteomics

Masanori Tachikawa
lab head
Masanori Tachikawa
contact affiliation	Tokushima University
dataset submitter

jPOST dataset URI

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