PXD041290

PXD041290 is an original dataset announced via ProteomeXchange.

Title	TMT-based quantitative proteomics in Rab35 cKO P0 hippocampus
Description	The hippocampi from the control (Rab35flox/+; Nestin-Cre) and Rab35 cKO mice groups at P0 (n = 5 biological replicates per genotype) were lysed in a buffer containing 6 M guanidine hydrochloride, 100 mM HEPES-NaOH (pH 8.0), 10 mM TCEP, and 40 mM chloroacetamide. The lysates were dissolved through heating and sonication, followed by centrifugation at 15,000 rpm for 15 min at 4°C. Proteins (100 μg each) were purified using methanol/chloroform precipitation and solubilized through the addition of 20 μL of 0.1% RapiGest SF (Waters) in 50 mM triethylammonium bicarbonate. After repeated sonication and vortexing, the proteins were digested with 1 μg of trypsin/Lys-C mix (Promega) for 16 h at 37°C. The peptide concentrations were determined using the Pierce quantitative colorimetric peptide assay (Thermo Fisher Scientific). Approximately 25 μg of peptides for each sample was labeled with 0.2 mg of TMT10-plex reagents (Thermo Fisher Scientific) for 1 h at 25°C. After the reaction was quenched with hydroxylamine, all the TMT-labeled samples were pooled, acidified with trifluoroacetic acid (TFA), and fractionated using the Pierce high pH reversed-phase peptide fractionation kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Nine fractions were collected using 5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, and 80% acetonitrile (ACN). 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 (1 μg each) 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 with a linear gradient from 4–20% ACN for min 0–180 and 20–32% ACN for min 180–220, followed by an increase to 80% ACN during min 220–230. The mass spectrometer was operated in a data-dependent acquisition mode with a top 15 MS/MS method. MS1 spectra were measured with a resolution of 70,000, an automatic gain control (AGC) target of 3e6, and a mass range of 375–1,400 m/z. HCD MS/MS spectra were acquired at a resolution of 35,000, an AGC target of 1e5, an isolation window of 0.4 m/z, a maximum injection time of 100 msec, and a normalized collision energy of 34. Dynamic exclusion was set to 30 sec. Raw data were directly analyzed against the SwissProt database restricted to M. musculus using Proteome Discoverer version 2.3 (Thermo Fisher Scientific) with Mascot search engine version 2.5 (Matrix Science) 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) TMT of lysine and peptide N-terminus and carbamidomethylation of cysteine as fixed modifications; and (e) oxidation of methionine as a variable modification. Peptides were filtered at a false-discovery rate (FDR) of 1% using the percolator node.
HostingRepository	jPOST
AnnounceDate	2023-04-07
AnnouncementXML	Submission_2023-04-23_19:24:11.828.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Hidetaka Kosako
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	S-carboxamidomethyl-L-cysteine; L-methionine sulfoxide; TMT6plex reporter+balance reagent N-acylated residue; TMT6plex reporter+balance reagent acylated N-terminal
Instrument	instrument

Revision	Datetime	Status	ChangeLog Entry
0	2023-04-02 20:49:26	ID requested
1	2023-04-06 15:02:10	announced
⏵ 2	2023-04-23 19:24:13	announced	2023-04-24: Updated PubMed.

Maejima I, Hara T, Tsukamoto S, Koizumi H, Kawauchi T, Akuzawa T, Hirai R, Kobayashi H, Isobe I, Emoto K, Kosako H, Sato K, RAB35 is required for murine hippocampal development and functions by regulating neuronal cell distribution. Commun Biol, 6(1):440(2023) [pubmed]

submitter keyword: TMT, Rab35, hippocampus

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

jPOST dataset URI

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