PXD053025

PXD053025 is an original dataset announced via ProteomeXchange.

Title	Proteome data of WT and ob/ob mice liver during starvation
Description	Ten-week-old wilt-type (WT) and ob/ob mice were starved for 0, 2, 4, 6, 8, 12, 16, and 24 hours and liver samples were collected. Total proteins extracted with methanol:chroloform:water (2.5:2.5:1) and subjected to the following proteomic analysis. For standardization among runs of measurement, proteins from frozen liver powder of Lys(6)-SILAC mice (Silantes) were also extracted with methanol/chloroform extraction. Protein concentration of the lysates was determined using the bicinchoninic acid (BCA) assay (23227, Thermo Fisher Scientific) and calibrated to 1 mg/mL. Then, 50 µg each of non-labeled and labeled lysates were mixed for peptide preparation. Cysteine residues were blocked by 2 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (Thermo Fisher Scientific) at 37°C for 30 min followed by alkylation with 10 mM 2-iodoacetamide (IAA) at room temperature for 30 min. The proteins were precipitated with acetone for 3 hours at −30°C and the resulting pellet was dispersed in 50 mM triethylammonium bicarbonate by ultrasonic treatment (three times for 30 s with intervals of 30 s) with a Bioruptor (Diagenode). The protein suspension was subjected to digestion with lysyl endopeptidase (Wako) for 16 h at 37°C. Resulting peptides were centrifuged at 15,000 × g for 15 min at 4°C and subjected to C18-StageTip purification. Peptides were resolved in 50 µL 3% acetonitrile (can)-0.1% foromic acid prior to MS analysis. All samples were analyzed with Q Exactive HF-X (Thermo Fisher Scientific) instrument equipped with an UltiMate 3000 RSLCnano LC System (Thermo Fisher Scientific) via a nano-electrospray source with a column oven set at 50°C (AMR Inc.). Peptides were directly injected onto a 75 μm × 25 cm PicoFrit emitter (New Objective, Woburn, MA, USA) packed in-house with C18 core-shell particles (CAPCELL CORE MP 2.7 μm, 160 Å material; Osaka Soda Co., Ltd., Osaka, Japan) with a linear gradient of 7–42% B for 76 min, 42-80% B for 5 min, and 80% B for 9 min at a flow rate of 100 nL/min, where A is 0.1% formic acid and B is 0.1% formic acid and 80% acetonitrile. MS data acquisition was performed in data-independent acquisition (DIA) mode. For quantification in the DIA mode, MS1 spectra were collected in the range of m/z 423-865 at 60,000 resolution to set an AGC target of 3 × 10^6. For MS2 spectra, collections were conducted in the range of > m/z 200 at 30,000 resolution to set an AGC target of 3 × 10^6. The isolation width was configured to 8 Th with stepped normalized collision energies of 20, and 23%. Through the optimization by Skyline 4.1, isolation window patterns in m/z 428-860 were used as window placements. In the DIA mode for a spectral library, the pool of all samples was analyzed by using the above measurements and the gas-phase fractionation method. In the gas-phase fraction method, six MS ranges (m/z 426-502, 498-574, 570-646, 642-718, 714-790, and 786-862) were used, and each was measured by overlapping DIA10. MS1 spectra were collected at 120,000 resolution to set an AGC target of 3 × 10^6. MS2 spectra were collected in the range of > m/z 200 at 60,000 resolution to set an AGC target of 3 × 10^6. The isolation width was set to 2 Th with stepped normalized collision energies of 20 and 23%. Isolation window patterns in the ranges of m/z 428-500, 500-572, 572-644, 644-716, 716-788, and 788-860 were used as window placements optimized by Skyline 4.1. The spectra library was generated by searching MS data in the library against the mouse Ensembl release 97 database using Scaffold DIA (Proteome Software Inc., Portland OR, USA). The parameters were as follows: experimental data search enzyme, LysC/P; maximum missed cleavage sites, 1; precursor mass tolerance, 6 ppm; fragment mass tolerance, 8 ppm; and static modification, cysteine carbamidomethylation. The threshold for identification of peptides was a peptide false discovery rate (FDR) < 1%. To identify peptides derived from Lys(6)-SILAC mice, a library of identified peptides with a mass increase 6.020129 Da on Lys was built and added to the library obtained above. All raw data was processed with DIA-NN (ver. 1.8) using library search mode using the following parameters: reannotation, Mus_musculus.GRCm38.97.pep.all.fa; modification, UniMod188 (6.020129), and peak-translation. Using the output from DIA-NN software, the SILAC ratios of all assigned fragment ion pairs were calculated, and the median of the SILAC ratios of each protein was used for protein quantification. Proteins assigned to a single ensemble gene ID were used in the following analyses. The SILAC ratios of all the proteins were normalized by the median of the SILAC ratio in each biological sample.Note that data at 0 hour were already public (PXD048481)
HostingRepository	jPOST
AnnounceDate	2025-05-20
AnnouncementXML	Submission_2025-05-19_20:18:01.821.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Keigo Morita
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	S-carboxamidomethyl-L-cysteine; 6x(13)C labeled L-lysine
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2024-06-11 16:42:14	ID requested
⏵ 1	2025-05-19 20:18:02	announced

Dataset with its publication pending

submitter keyword: proteome, WT, ob/ob, mouse, liver, starvation, time-course

Shinya Kuroda
lab head
Keigo Morita
contact affiliation	University of Tokyo
dataset submitter

jPOST dataset URI

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