Dataset Information

Proteome analysis of mouse liver and skeletal muscle

ABSTRACT: The liver and skeletal muscle were collected from 10-week-old wild-type and ob/ob mice fasted for 16 hours. Tissues were frozen immediately and lysed with 0.5 mL of a solution containing 50 mM Tris-HCl pH8.8, 2%SDS, and 7 M urea, and then subjected to ultrasonic treatment with a Bioruptor (Digaenode). The samples were diluted with an equal volume of water and centrifuged at 15,000 g for 15 min at 4°C to remove insoluble fraction. The protein concentration of the lysates was determined with the bicinchoninic acid assay (Thermo Fisher Scientific) and adjusted to 1 mg/mL. Cysteine residues were blocked by incubation of the samples with 2 mM tris(2-carboxyethyl)phosphine hydrochloride (Thermo Fisher Scientific) for 30 min at 37 °C followed by alkylation with 10 mM 2-iodoacetamide for 30 min at room temperature. The proteins (200 µg) were precipitated with acetone for 3 h 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 prior to MS analysis

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Shinya Kuroda

PROVIDER: PXD020656 | JPOST Repository | Wed Mar 03 00:00:00 GMT 2021

REPOSITORIES: jPOST

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Dataset's files

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			Action	DRS
	20171209_iBAQ_Muscle_600ng_01.raw	Raw
	20171209_iBAQ_Muscle_600ng_02.raw	Raw
	20171209_iBAQ_Muscle_600ng_03.raw	Raw
	20171209_iBAQ_Muscle_600ng_04.raw	Raw
	20171209_iBAQ_Muscle_600ng_05.raw	Raw

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Publications

Trans-omic analysis reveals obesity-associated dysregulation of inter-organ metabolic cycles between the liver and skeletal muscle.

Egami Riku R Kokaji Toshiya T Hatano Atsushi A Yugi Katsuyuki K Eto Miki M Morita Keigo K Ohno Satoshi S Fujii Masashi M Hironaka Ken-Ichi KI Uematsu Saori S Terakawa Akira A Bai Yunfan Y Pan Yifei Y Tsuchiya Takaho T Ozaki Haruka H Inoue Hiroshi H Uda Shinsuke S Kubota Hiroyuki H Suzuki Yutaka Y Matsumoto Masaki M Nakayama Keiichi I KI Hirayama Akiyoshi A Soga Tomoyoshi T Kuroda Shinya S

iScience 20210220 3

Systemic metabolic homeostasis is regulated by inter-organ metabolic cycles involving multiple organs. Obesity impairs inter-organ metabolic cycles, resulting in metabolic diseases. The systemic landscape of dysregulated inter-organ metabolic cycles in obesity has yet to be explored. Here, we measured the transcriptome, proteome, and metabolome in the liver and skeletal muscle and the metabolome in blood of fasted wild-type and leptin-deficient obese (<i>ob</i>/<i>ob</i>) mice, identifying compo ...[more]

PMID: 33748705

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Project description:Human Umbilical Vein Endothelial Cells pooled from several donors, were purchased from Lonza and cultured in FBS reduced Endopan 3 media. HUVEC cells of passages 5-10 were used for experiments. MPO treatment was performed with protein purified from human blood (Planta) and at 1 μg/ml final concentration. Immunoprecipitation was performed from isolated nuclei (after 8 hours MPO treatment), non-treated cells were used as negative control. For immunoprecipitation antibody against MPO (Calbiochem, 475915) was used. Cell nuclei were isolated by incubating cells for 15 min on ice in NIB buffer (15 mM Tris-HCL pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 250 mM sucrose) containing 0.3% NP-40. Nuclei were pelleted for 5 min 800 × g at 4 °C, washed twice in the same buffer, lysed for 10 min on ice in IP buffer (150 mM LiCl, 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.5% Empigen) freshly supplemented with 2 mM sodium vanadate, 1× protease inhibitor cocktail (Roche), PMSF (10 µl), 0,5 mM DTT, and 50 units Benzonase per ml of IP buffer, before preclearing cell debris by centrifugation at >15,000 × g at 4 °C. Finally, 1 mg of the lysate was incubated with anti-MPO antisera overnight at 4 °C. Magnetic beads (Active Motif) were then washed once with 1× PBS-Tween and combined with the antibody-lysate mixture. Following a 2-h incubation at 4 °C, beads were separated on a magnetic rack and washed 5×, 5 min each in wash buffer (150 mM KCl, 5 mM MgCl2, 50 mM Tris-HCl pH 7.5, 0.5% NP-40) and another two times in wash buffer without NP-40. Captured proteins were predigested and eluted from the beads using digestion buffer (2 M Urea, 50 mM Tris-HCl pH 7.5, 1 mM DTT) supplemented with trypsin and eluted from the beads with elution buffer (2 M Urea, 50 mM Tris-HCl pH 7.5, 5 mM chloroacetamide) supplemented with trypsin and LysC, before subjected to mass-spectrometry on a Q-Exactive Plus Orbitrap platform coupled to an EASY nLC (Thermo Scientific). Peptides were loaded in solvent A (0.1% formic acid in water) onto an in-house packed analytical column (50 cm length, 75 µm I.D., filled with 2.7 µm Poroshell EC120 C1; Agilent)