PXD053026

PXD053026 is an original dataset announced via ProteomeXchange.

Title	Phosphoproteome data of WT and ob/ob mice liver during starvation
Description	Ten-week-old male mice (WT and ob/ob) were starved for 0, 2, 4, 6, 8, 12, 16, and 24 hours and liver samples were collected. Total proteins extracted during sample preparation of metabolomic analysis were subjected to the following phosphoproteomic analysis. As described above, 2 mg of protein solution was subjected to reduction and alkylation by TCEP and IAA, respectively. Then, proteins were precipitated in three volumes of ice-cold acetone and centrifuged at 16,000 × g for 2 h. The pellet was rinsed with 90% acetone followed by digestion with 30 µg of trypsin in 600 µl of 100 mM ammonium bicarbonate for 16 h. Thirty-five µl of 10% trifluoroacetic acid (TFA) and 450 µl of ACN were added to the protein digests (1 mg/300 µl). The mixture was centrifuged at 16,000 × g and the supernatant was collected into a new tube. Phosphopeptides were enriched using Fe3+-IMAC104. NTA-agarose resin (1 mL) (Qiagen) was washed twice with 3 ml of distilled water and 2 ml of 100 mM FeCl3 in 0.1% acetic acid was added. Following a wash with 2 ml of 0.1% TFA, 2% ACN and 0.1% TFA, 60% ACN, the resin was stored in 1 ml of 0.1% TFA, 60% ACN (Fe3+-IMAC agarose resin). C18-StageTip (3M) packed with 75 µl of Fe3+-IMAC agarose resin was equilibrated with 200 µl of 0.1% TFA, 60% ACN, then the protein digests were loaded onto the IMAC/C18-StageTip and the loaded system was washed twice with 200 µL of 0.1% TFA, 60% ACN. Following equilibration with 200 µl of 0.1% TFA, 2% ACN, 200 µl of 1% inorganic phosphate was added to elute phosphopeptides from the Fe3+-IMAC agarose resin and bind them to the C18-StageTip. Following a wash with 200 µL of 0.1% TFA, 2% ACN, the peptides were eluted with 0.1% TFA, 60%ACN from the IMAC/C18-StageTip. The resulting peptides were dried, reconstituted in 3% ACN with 0.1% formic acid, and subjected to MS analysis. All samples were analyzed with Orbitrap Exploris 480 MS (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 60°C (AMR Inc.). Peptides were directly applied onto a 75 μm × 120 mm column (Nikkyo Technos Co.,Ltd) with a linear gradient of 5–32% B for 70 min and 32-65% B for 10 min at a speed of 200 nL/min, where A is 0.1% formic acid and B is 0.1% formic acid and 80% acetonitrile. MS data were acquired in overlapping data-independent acquisition (DIA) mode. For quantification in the DIA mode, MS1 spectra were collected in the range of m/z 390-1010 at 30,000 resolution to set an AGC target of 3 × 10^6. MS2 spectra were collected in the range of > m/z 200 at 30,000 resolution to set an AGC target of 3 × 10^6, and stepped normalized collision energies of 22, 26, and 30%. The isolation width for MS2 was set to 10 Th and overlapping window patterns in m/z 400-1000 were used window placements optimized by Skyline v4.1. In the DDA-MS mode for a spectral library, the pool of all samples was analyzed by using the gas-phase fractionation method with MS ranges of m/z 395-555, 545-705, 695-1005, and 390-1010. MS1 spectra were collected at 120,000 resolution to set an AGC target of 3 × 10^6. Within the MS ranges of m/z 395-555, 545-705, 695-1005, the 20 most intense ions with charge states of 2+ to 5+ more than 5.0 × 10^3 were fragmented by HCD with normalized collision energies of 22, 26, and 30%, and MS2 spectra were collected in the range of > m/z 200 at 60,000 resolution to set an AGC target of 5 × 10^5. Within the MS ranges of m/z 390-1010, the 40 most intense ions with charge states of 2+ to 5+ that exceeded 5.0 × 10^3 were fragmented by HCD with normalized collision energies of 22, 26, and 30%, and MS2 spectra were collected in the range of > m/z 200 at 30,000 resolution to set an AGC target of 2 × 10^5. The spectra library was built by searching MS data in the library against the mouse Ensembl database (GRCm38/mm10, Ensembl release 97) using Proteome Discoverer v2.3 (Thermo Fisher Scientific). The setting parameters were as follows: Fragmentation, HCD; Precursor Tolerance, 8 ppm; Fragment Tolerance, 0.02 Da; Digestion Enzyme, Trypsin; Max Missed Cleavages, 1; Variable Modification, Phospho [S, T, Y]; Fixed Modification, Carbamidomethylation [C]; Site Probability Threshold, 75; Peptide FDR, < 1%. Unique phosphopeptides were subjected to the following analyses. To determine phosphorylation at each phosphosite, the intensities of all signals of quantified phosphopeptides that include the phosphosite of interest were summed.Note that data at 0 hour were already public (PXD048480)
HostingRepository	jPOST
AnnounceDate	2025-05-20
AnnouncementXML	Submission_2025-05-19_20:26:25.428.xml
DigitalObjectIdentifier
ReviewLevel	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; O-phospho-L-serine; O-phospho-L-threonine; O4'-phospho-L-tyrosine
Instrument	LTQ Orbitrap

Revision	Datetime	Status	ChangeLog Entry
0	2024-06-11 16:47:46	ID requested
1	2025-05-19 20:18:59	announced
⏵ 2	2025-05-19 20:26:26	announced	2025-05-20: Updated PubMed.

Morita K, Hatano A, Kokaji T, Sugimoto H, Tsuchiya T, Ozaki H, Egami R, Li D, Terakawa A, Ohno S, Inoue H, Inaba Y, Suzuki Y, Matsumoto M, Takahashi M, Izumi Y, Bamba T, Hirayama A, Soga T, Kuroda S, Structural robustness and temporal vulnerability of the starvation-responsive metabolic network in healthy and obese mouse liver. Sci Signal, 18(883):eads2547(2025) [pubmed]

submitter keyword: phosphoproteome, 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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