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Regulation of cold-induced thermogenesis by the stress granule protein FAM195A

ABSTRACT: 8 fractions for the TMT with file number of 748186_F1 to F8 was used for this section. Protein lysates were extracted from 3 WT and 3 FAM195A KO mice using RIPA lysis buffer. Protein concentration was determined and 25 ug of protein were processed. After trypsin digestion samples were then labelled with TMT reagent. A reverse-phase fractionation spin column was used according to the manufacturers directions to fractionate the sample into 8 fractions. Fractions were injected onto an Orbitrap Fusion Lumos mass spectrometer coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system. The mass spectrometer operated in positive ion mode with a source voltage of 1.6 kV and an ion transfer tube temperature of 275 C. MS scans were acquired at 120,000 resolution in the Orbitrap and top speed mode was used for SPS-MS3 analysis with a cycle time of 2.5 sec. MS2 was performed with CID with a collision energy of 35%. The top 10 fragments were selected for MS3 fragmentation using HCD, with a collision energy of 55%. Raw MS data files were analyzed using Proteome Discoverer v2.2 (Thermo), with peptide identification performed using Sequest HT searching against themouse protein database from UniProt. Fragment and precursor tolerances of 10 ppm and 0.6 Da were specified, and three missed cleavages were allowed. Carbamidomethylation of Cys and TMT labelling of N-termini and Lys side-chains were set as a fixed modification, with oxidation of Met set as a variable modification. Description for sample PCF-JC-2191-07-2020 ( 783251-2) and PCF-JC-2227-07-2020 (787223-4) Differentiated brown adipocytes stably expressing FAM195A-miniTurbo were supplemented with 50 uM biotin (2 h) or left untreated. Cell lysates were extracted in 6 M urea RIPA. Biotinylated proteins were obtained after incubation of lysates with streptavidin beads. Samples were run for 1 cm in a protein gel. The area of the gel containing proteins was submitted for analysis. Gel band samples were digested overnight with trypsin following reduction and alkylation with DTT and iodoacetamide. The samples then underwent solid-phase extraction cleanup with an Oasis HLB plate (Waters) and the resulting samples were injected onto an Orbitrap Fusion Lumos mass spectrometer coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system. Samples were injected onto a 75 um i.d., 75-cm long EasySpray column and eluted with a gradient from 0-28% buffer B over 90 min. Buffer A contained 2% (v/v) ACN and 0.1% formic acid in water, and buffer B contained 80% (v/v) ACN, 10% (v/v) trifluoroethanol, and 0.1% formic acid in water. The mass spectrometer operated in positive ion mode with a source voltage of 1.8 kV and an ion transfer tube temperature of 275 C. MS scans were acquired at 120,000 resolution in the Orbitrap and up to 10 MS/MS spectra were obtained in the ion trap for each full spectrum acquired using higher-energy collisional dissociation (HCD) for ions with charges 2-7. Dynamic exclusion was set for 25 sec after an ion was selected for fragmentation. The Proteomics Core Facility at UTSW performed all proteomic analysis reported.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Eric N. Olson

PROVIDER: MSV000086711 | MassIVE | Fri Jan 15 21:10:00 GMT 2021

REPOSITORIES: MassIVE

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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:Sample description: Anti-PD-L1 co-immunoprecipitation samples from cultured wild-type, PD-L1-VavCre or PD-L1-LysMCre bone marrow cells. Samples are: KO-VavCre-1 (775112) v.s. WT-1 (775113); KO-LysMCre-2 (775114) v.s. WT-2 (775115). Among these samples, KO-VavCre-1 and KO-LysMCre-2 are the respective blank controls (from knockout mice). Three 10-cm plates of bone marrow cells from each genotype (wild-type or PD-L1-VavCre, and wild-type or PD-L1-LysMCre) of 2-month-old male littermate mice at a density of 2.8 * 107 cells/plate were cultured in the presence of 40 ng/mL of mouse M-CSF for 3 days. Cells were washed with PBS and resuspend in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM PMSF, 1x protease inhibitor cocktail) for 4 hours at 4C. The lysate was centrifuged at 12,000 g for 10 min. The supernatant was mixed with 2 micro G of anti-PD-L1 antibody and incubated for overnight at 4C. Protein A/G agarose (Santa Cruz) was added and incubated for 2 hours at 4C. Agarose was washed with lysis buffer for four times. The samples were separated in SDS-PAGE gel (10-mm length per lane). Coomassie blue stained gel slices were analyzed by mass spectrometry. Samples were digested overnight with trypsin (Pierce) following reduction and alkylation with DTT and iodoacetamide (Sigma-Aldrich). The samples then underwent solid-phase extraction cleanup with an Oasis HLB plate (Waters) and were subsequently dried and reconstituted into 10 microL of 2% ACN, 0.1% TFA. 2 microL of these samples were injected onto a QExactive HF mass spectrometer coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system. Samples were injected onto a 75 micro M i.d., 15-cm long EasySpray column (Thermo) and eluted with a gradient from 0-28% buffer B over 90 min with a flow rate of 250 nL/min. Buffer A contained 2% (v/v) ACN and 0.1% formic acid in water, and buffer B contained 80% (v/v) ACN, 10% (v/v) trifluoroethanol, and 0.1% formic acid in water. The mass spectrometer operated in positive ion mode with a source voltage of 2.2 kV and an ion transfer tube temperature of 275 C. MS scans were acquired at 120,000 resolution in the Orbitrap and up to 20 MS/MS spectra were obtained for each full spectrum acquired using higher-energy collisional dissociation (HCD) for ions with charges 2-8. Dynamic exclusion was set for 20 second after an ion was selected for fragmentation.

Project description:Ammonium hydroxide and pyrrolidine eluents were dried (SpeedVac) and reconstituted in 25 ul sample loading buffer (0.1% TFA/2% acetonitrile). Eluent from phosphotyrosine immunoprecipitation was dried and reconstituted in 35 ul of the loading buffer. An LTQ Orbitrap XL (ThermoFisher) in-line with a Paradigm MS2 HPLC (Michrom bioresources) was employed for acquiring high-resolution MS and MS/MS data. Ten microliters of the phospho-enriched peptides were loaded onto a sample trap (Captrap, Bruker-Michrom) in-line with a nano-capillary column (Picofrit, 75 um i.d.x 15 um tip, New Objective) packed in-house with 10 cm of MAGIC AQ C18 reverse phase material (Michrom Bioresource). Two different gradient programs, one each for MOAC and phosphotyrosine immunoprecipitation samples, were used for peptide elution. For MOAC samples, a gradient of 5-40% buffer B (95% acetonitrile/1% acetic acid) in 135 min and 5 min wash with 100% buffer B followed by 30 min of re-equilibration with buffer A (2% acetonitrile/1% acetic acid) was used. For phosphotyrosine immunoprecipitation samples, which were a much less complex mixture of peptides, 5-40% gradient with buffer B was achieved in 75 min followed by 5 min wash with buffer B and 30 min re-equilibration. Flow rate was ~0.3 ul/min. Peptides were directly introduced into the mass spectrometer using a nano-spray source. Orbitrap was set to collect 1 MS scan between 400-2000 m/z (resolution of 30,000 @ 400 m/z) in orbitrap followed by data dependent CID spectra on top 9 ions in LTQ (normalized collision energy ~35%). Dynamic exclusion was set to 2 MS/MS acquisitions followed by exclusion of the same precursor ion for 2 min. Maximum ion injection times were set to 300 ms for MS and 100 ms for MS/MS. Automatic Gain Control (AGC) was set to 1xe6 for MS and 5000 for MS/MS. Charge state screening was enabled to discard +1 and unassigned charge states. Technical duplicate data for each of the MOAC elutions (ammonium hydroxide and pyrrolidine) and triplicate data for the phosphotyrosine immunoprecipitation samples were acquired. RAW files were converted to mzXML using msconvert and searched against the Swissprot Human protein database (2013-Jan release) appended with common proteomics contaminants and reverse sequences as decoys. Searches were performed with X!Tandem (version 2010.10.01.1) using the k-score plugin. For all searches the following search parameters were used: Parent monoisotopic mass error of 50 ppm; fragment ion error of 0.8 Daltons; allowing for up to 2 missed tryptic cleavages. Variable modifications were oxidation of Methionine (+15.9949@M), carbamidomethylation of Cysteine (+57.0214@C), and phosphorylation of Serine, Threonine, and Tyrosine (+79.9663@[STY]). The search results were then post-processed using PeptideProphet and ProteinProphet.

Project description:Proteomic analysis of chick vestibualr hair bundles purified using the twist-off method. See Description.doc. Utricle hair bundles were purified from E20–21 chicks using the twist-off method (Gillespie & Hudspeth J Cell Biol 112, 625-640, 1991; Shin et al. Neuron 53, 371-386, 2007). NuPAGE LDS sample buffer (Invitrogen) with 50 mM dithiothreitol was added to a combined final volume of 80 µl per 100 utricles' worth of bundles; samples were heated to 70°C for 15 min. Epithelial proteins were similarly solubilized with sample buffer. Proteins were separated by running ~1 cm into a NuPAGE 4–12% Bis-Tris gel (1.5 mm x 10 well; one or two lanes per bundle sample); gels were rinsed with water, then stained with Imperial Protein Stain (Thermo Scientific). The 1 cm of gel with separated proteins was manually sliced into six pieces. Gel pieces were transferred to siliconized tubes and washed and destained in 200 µl 50% methanol overnight. The gel pieces were dehydrated in acetonitrile, rehydrated in 30 µl of 10 mM dithiothreitol in 0.1 M ammonium bicarbonate and reduced at room temperature for 0.5 h. The DTT solution was removed and the sample alkylated in 30 µl 50 mM iodoacetamide in 0.1 M ammonium bicarbonate at room temperature for 0.5 h. The reagent was removed and the gel pieces dehydrated in 100 µl acetonitrile. The acetonitrile was removed and the gel pieces rehydrated in 100 µl 0.1 M ammonium bicarbonate. The pieces were dehydrated in 100 µl acetonitrile, the acetonitrile removed and the pieces completely dried by vacuum centrifugation. The gel pieces were rehydrated in 20 ng/µl trypsin (Sigma-Aldrich T6567 proteomics grade, from porcine pancreas, dimethylated) in 50 mM ammonium bicarbonate on ice for 10 min. Any excess enzyme solution was removed and 20 µl 50 mM ammonium bicarbonate added. The sample was digested overnight at 37°C and the peptides formed extracted from the polyacrylamide in two 30 µl aliquots of 50% acetonitrile/5% formic acid. These extracts were combined and evaporated to 15 µl for MS analysis. For the gel slice immediately adjacent to the agarose, peptides were purified away from interfering polymers by SCX. A single experiment's worth of hair bundles or epithelium was analyzed by six LC-MS/MS runs, corresponding to the six gel pieces. The LC-MS/MS system consisted of a Thermo Electron Orbitrap Velos ETD mass spectrometer system with a Protana nanospray ion source, which was interfaced to a reversed-phase capillary column of 8 cm length x 75 µm internal diameter, self-packed with Phenomenex C18 Jupiter of 10 µm particle size. An extract aliquot (7.5 µl) was injected and peptides eluted from the column by an acetonitrile/0.1 M acetic acid gradient at a flow rate of 0.5 µl/min over 1.2 hr. The nanospray ion source was operated at 2.5 kV. The digest was analyzed using the data-dependent capability of the instrument, acquiring—in sequential scans—a single full scan mass spectrum in the Orbitrap detector at 60,000 resolution to determine peptide molecular weights, and 20 product-ion spectra in the ion trap to determine amino acid sequence. MaxQuant version 1.2.2.5 software was used for protein identification and quantitation. The default contaminants file associated with the MaxQuant download was edited to remove entries known to be present in hair bundles (e.g., actin) and to add additional impurities that entered the bundle-purification workflow (keratins, hemoglobins, egg white components). Mass spectrometry data were searched against Ensembl version 66 (released February, 2012) using Andromeda; the Ensembl FASTA file was edited by replacing several sequences with longer or full-length sequences, including actin gamma 1 (NP_001007825.1), actin beta (NP_990849.1), fascin 1 (NP_001171603), fascin 2 (NP_001171209), ATP synthase beta (NP_001026562.1), peptidylprolyl isomerase A (NP_001159798.1), calbindin 2 (NP_990647.1), PDZD7 (XP_003641537.1), espin (XP_417532.3), and CACNA2D2 (XP_427707.3). Protein identifications were reported with an FDR of 1%. If a set of peptides for a protein was identical to or completely contained within that of another protein, MaxQuant groups those proteins together ("redundant groups"); the entry with the largest number of peptides was used to identify the redundant group. Redundant groups that shared more than 20% of their identified peptides were further grouped in our analysis ("shared-peptide groups"); the entry with the greatest intensity associated with it was used to identify the shared-peptide group.

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Regulation of cold-induced thermogenesis by the stress granule protein FAM195A

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