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Phosphoproteome analysis of mouse liver at ad libitum feeding

ABSTRACT: The liver samples were collected from ad libitum feeding 10-week-old wild-type and ob/ob mice. Tissues were lysed with 0.5 mL of a solution containing 50 mM Tris-HCl pH8.8, 2% sodium dodecyl sulfate (SDS), and 7 M urea, and then homogenized by ultrasonication using a Bioruptor (Digaenode, Denville, NJ, USA). After dilution with equal amount of water, the samples were centrifuged at 15,000 g for 15 min at 4°C to exclude the insoluble fractions. Bicinchoninic acid (BCA) assay (Thermo Fisher Scientific) was used to quantify the protein concentration of the lysates, and lysate concentration was adjusted to 1 mg/mL. Cysteine residues were blocked using 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. Proteins were precipitated by addition of acetone. The resulting protein pellet was digested with trypsin in 100 mM ammonium bicarobonate. To enrich the phosphopeptides, the resulting peptides (1 mg) were subjected to Fe3+-IMAC. All samples were analyzed with Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) equipped with the 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 injected onto a 75 μm × 120 mm column (Nikkyo Technos Co.,Ltd., Tokyo, Japan) with a linear gradient of 5–32% B for 70 min and 32–65% B for 10 min at a flow rate of 200 nL/min, where A is 0.1% formic acid and B is 0.1% formic acid and 80% acetonitrile.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Shinya Kuroda

PROVIDER: PXD048480 | JPOST Repository | Fri Jan 12 00:00:00 GMT 2024

REPOSITORIES: jPOST

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Project description:Glands were frozen at -80 ºC immediately after being retrieved and stored under this condition until initiating the proteomics analysis protocol. Glands were washed with cold PBS and immersed in 120 l of homogenization buffer (50 mM Tris-HCl, pH 6.8, 10 mM DTT, 4% (w/v) SDS). Glands were boiled for 5 min, incubated overnight at 4 ºC and centrifuged at 4 ºC and 13000 rpm for 10 min. The whole gland proteome of each gland was concentrated as described elsewhere (Bonzon-Kulichenko, F. Garcia-Marques, M. Trevisan-Herraz and J. Vazquez, Journal of Proteome Research, 2015, 14, 700-710 (DOI:10.1021/pr5007284).Briefly, samples were loaded into an SDS-PAGE gel (0.5 mm-thick, 4% stacking, and 10% resolving) and the electrophoresis process was stopped when the front entered 2 mm into the resolving gel. The unseparated protein bands were then visualized by Coomassie staining, excised and digested at 37 ºC overnight with 500 l of 25 g/l chymotrypsin (Promega) in 100 mM Tris-HCl, pH 7.8 containing 10 mM CaCl2. The cleaved peptides were extracted by incubation for 2 h in 100 mM Tris-HCl, pH 7.8 on shaking, acidized with 1% (v/v) trifluoroacetic acid, desalted onto C18 cartridges (Oasis, Waters Corporation, Milford, MA, USA), and dried down.The analysis of the samples proceeded through liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Easy nLC 1000 nano-HPLC coupled to a Q-Exactive Orbitrap mass spectrometer (Themo Scientific). Peptides were injected onto a C18 reverse-phase precolumn (Acclaim PepMap100, 75-m i.d., 3-m particle size, 2-cm length, Thermo Scientific), and a reverse-phase analytical column (Acclaim PepMap 100, 75-m, i.d., 3-m particle size, 50-cm length, Thermo Scientific) in buffer A [0.1% formic acid (v/v)] and eluted with a 60 min linear gradient of buffer B [90% acetonitrile, 0.1% formic acid (v/v)], at 200 nL/min. Mass spectrometry (MS) runs consisted of 70000 FT-resolution spectra in the 390-1200 m/z wide range, followed by data-dependent MS/MS spectra of the 15 most intense parent ions acquired along the chromatographic run. High-energy collisional (HCD) fragmentation was performed at 27% of normalized collision energy and the isolation window for the parent ion mass was established at 2 Da.

Project description:Proteome quantification using TMT reagents and off-line basic peptide fractionation was done as described previously in Grand, 2021, with the exception that the isolated nuclei were used as a starting material rather than whole cells to enable detection of transcription factors which are generally less abundant proteins in the cell, and that nine channels from a TMTpro16 reagent kit were used. Briefly, mESCs, NGN2 24h induced cells and MyoD1 24h induced cells were harvested in triplicates of 10 million per sample. Nuclei were isolated by suspending in 4 ml of nuclear extraction buffer (10 mM HEPES pH 7.9, 10 mM KCl, 10 mM EDTA, 0.5% NP-40, 1 mM DTT and complete protease inhibitor (Roche)) and incubating on ice for 10 min with vortexing every 2 minutes. Nuclei were collected by centrifugation (800g, 10 min, 4 °C), washed with ice cold PBS and suspended in 1% sodium deoxycholate in 50 mM HEPES buffer (pH 8.5). The nuclear lysate was disrupted with sonication. Proteins were reduced, alkylated and Lys-C/trypsin digested. Fifty micrograms of peptides from each biological replicate were labelled using channels 128C-131C from a TMT 16plex isobaric labelling reagents kit (Lot# VJ313476, Thermo Fisher Scientific). Dried TMT-labeled peptides were dissolved in 20 μL of 10 mM ammonium formate (pH 10, high pH Buffer A), and high-pH reversed-phase chromatography was employed using an Agilent 1100 HPLC system with an autosampler, a YMC Triart C18 0.5 x 250 mm column, and a fraction collector. The peptides were separated using the following binary buffer system: high-pH Buffer A (20 mM ammonium formate in water, pH 10) and high-pH Buffer B (20 mM ammonium formate pH 10 in 90% acetonitrile) at a flow rate of 12 μL/minute. The gradient duration was a total of 115 min, with mobile phase compositions in %B as follows: 0-5 min at 2%-15%, 5-15 min at 15%-25%, 15-80 min at 25%-45%, 80-85 min at 45%-65%, 85-95 min at 65%-100%, 95-100 min at 100%, 100-101 min at 100%-2%, and 101-115 min at 2%, resulting in 48 fractions after sample concatenation. Samples were acidified with 1% TFA and dried in a speed vac. Peptides reconstituted in 2% acetonitrile, 0.1% formic acid were on-line separated on a C18 50 cm μPAC column using a EASYnLC-1000 system (all Thermo Fisher Scientific) mounted on a Digital PicoView nanospray source (New Objective), connected to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Peptide elution was achieved using a linear gradient of increasing concentration of acetonitrile in 0.1% formic acid at a flow rate of 500 nl/min: 0-2 min: 2%, 2-8 min: 2-6%, 8-89 min 6-22%, 89-107 min: 22-40%, 107-111 min: 40-80%, 111-115 min: 80% (washing), 115-116 min: 80-2%, 116-120 min: 2% (equilibration). For MS data acquisition in a data-dependent mode, ions for survey MS1 scans were recorded in profile mode in the Orbitrap detector at a resolution of 120,000 in the range of 400-1400 m/z every 3 seconds for a maximum of 100 ms to reach the normalized AGC target of 50%. The most abundant precursors were selected in the quadrupole within an isolation window of 0.7 m/z for MS2 HCD fragmentation based on advanced peak determination, monoisotopic peak determination set to peptides, within charge states 2-8, dynamic exclusion of 60 s within +/-10 ppm tolerance for isotopes and different charge states, with a minimum intensity of 5e4, with normalized collision energy 34% for a maximum injection time of 200 ms to reach the normalized AGC target of 80%. MS2 HCD spectra were and recorded in the “normal” range, starting at 100 m/z at 50,000 resolution in the Orbitrap analyzer in centroid mode.

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)

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Phosphoproteome analysis of mouse liver at ad libitum feeding

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