Beta-cell Galphas signaling is critical for physiological and pharmacological enhancement of insulin secretion
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Exploris 480
ORGANISM(S): Mus Musculus (ncbitaxon:10090)
SUBMITTER: 
Jonathan Campbell
PROVIDER: MSV000097882 | MassIVE | Tue May 13 17:49:00 BST 2025
SECONDARY ACCESSION(S): PXD063902
REPOSITORIES: MassIVE
Similar Datasets
Project description:Peptides were labeled with TMT reagents based on the manufacturer’s instructions (Thermo Fisher Scientifific). To quantify six samples, each aliquot (100 µg of peptide equivalent) was reacted with one tube of TMT reagent. After the sample was dissolved in 100 µL of 0.05 M tetraethylammonium bromide (TEAB) solution, pH 8.5, the TMT reagent was dissolved in 41 µL of anhydrous acetonitrile. The mixture was incubated at room temperature for 1 hour.Then, 8 µL of 5% hydroxylamine was added to the sample and incubated for 15 min to quench the reaction. The 6-plex labeled samples were pooled together and lyophilized. The TMT-labeled peptides mixture was fractionated using a Waters XBridge BEH130 column (C18, 3.5 µm, 2.1 × 150 mm) on an Agilent 1290 high-performance liquid chromatography(HPLC) operating at 0.3 mL/min.
2020-05-16 | PXD019233 |
Project description:We've collected bulk RNA-Seq and TMT-labeled proteomic data of aorta, brain, heart, kidney, liver, lung, muscle (gastrocnemius muscle), and skin of 6-, 15-, 24-, and 30-months old male C57BL/6J mice. The tissues used for transcriptomic analysis and proteomic analysis were collected from the same mice. All analyses were conducted with 4 biological replicates.
2024-09-18 | GSE225576 | GEO
Project description:Heart tissue was lysed by sonication in 5% SDS (w/v) in 50 mM TEAB (pH 8.5) 450 microg of each sample was reduced with 10 mM DTT at 80 degC for 10 min and alkylated with 25 mM iodoacetamide in the dark for 30 min at room temperature. SDS was removed, and samples were digested with 20 microg of Sequencing Grade Modified Trypsin (Promega) using an S-trap mini device (Protifi) at 47 degC for 2 h. Lyophilized peptides were reconstituted in 100 microl of 200 mM TEAB buffer and labeled with 41 TMT10 reagents (lot# UL292365). After 2 h, reactions were quenched with hydroxylamine and combined.
TMT-labeled peptides were fractionated by high pH reversed-phase (HPRP) chromatography. Briefly, peptides were reconstituted in 400 microl of 20 mM ammonium formate, and 125 microl was fractionated using a 2.1 mm x 5 cm BEH C18 column (Waters) and Waters ACQUITY iClass UPLC. Separations utilized a flow rate of 0.4 ml/min and column temperature of 55 degC, and mobile phases consisted of 20 mM ammonium formate, pH 10 (MPA) and neat MeCN (MPB). Separations used a gradient as follows: 0 min, 3% B; 3 min, 7% B; 50 min, 50% B, 51 min, 90% B; 55 min, 90% B; 56 min, 3% B; 60 min, 3% B. Forty-eight equal fractions were collected over 52 minutes into a 96-well plate containing 10 microl of 20% TFA per well. This analysis was repeated 2 times total for fractionation of 2 mg peptides. The first 3 fractions of each injection were excluded, and the remaining samples were re-concatenated into 12 fractions. Approximately 5 microg of each fraction was separately aliquoted for analysis of the unenriched proteome, and samples were lyophilized.
Phosphopeptide enrichment used 200 microg of each of the 12 HPRP fractions was reconstituted in 80% MeCN/1% TFA containing 1M glycolic acid (buffer A), phosphopeptides were enriched using GL Sciences p10 TiO tips. After loading, tips were washed twice with buffer A followed by 2 times with 80% MeCN/1% TFA before elution with 20% MeCN/5% aqueous ammonia. After addition of neat formic acid, samples were lyophilized. Finally, peptides were desalted using C18 Stage Tips, lyophilized and reconstituted in 12 microl of 10 mM citrate in 1% TFA/2% MeCN. The unenriched fractions were reconstituted in 10 microl of 1% TFA/2% MeCN.
1D-LC-MS/MS was performed on 4.5 microl of each of the phospho-enriched fractions or on 0.75 microg of unenriched fractions. Samples were analyzed using a nanoACQUITY UPLC system (Waters) coupled to a coupled to a Exploris 480 high resolution accurate mass tandem mass spectrometer (Thermo) via a nanoelectrospray ionization source and FAIMS Pro Interface. Samples were first trapped on a Symmetry C18 180 microm x 20 mm trapping column (5 microl/min at 99.9/0.1 v/v H2O/MeCN) followed by an analytical separation using a 1.7 microm Acquity HSS T3 C18 75 microm x 250 mm column (Waters) with a 90 min gradient of 5 to 30% MeCN with 0.1% formic acid at a flow rate of 400 nl/min and column temperature of 55 degC. Data collection on was performed in data-dependent acquisition (DDA) mode with 3 FAIMS compensation voltages (-40, -60 and -80). Each CV used a 60,000 resolution full MS scan from m/z 375 to 1600 with a normalized AGC target of 300%, peptide monoisotopic peak determination, an intensity threshold of 5E3 ions, precursor fit of 70% with 0.7 m/z fit window, charge state of 2-5 and 40 s dynamic exclusion. MS/MS used a top6 method with 30,000 resolution and TurboTMT enabled, an isolation width of 0.7 m/z, NCE of 36, AGC target of 300% and maximum IT of 120 ms. Advanced precursor determination was enabled. The analysis of unenriched samples used a similar method except that a 1 s total scan time was used for each CV and MS/MS used an auto IT.
| MSV000086199 | MassIVE
Project description:We used whole mouse genome microarrays to the effects of γ-TmT on global gene expression in the prostate TRAMP and age-matched C57BL/6 mice were administered either 600 mg/kg of γ-TmT or a control vehicle via oral gavage. Twelve hours after dosing, prostate tissues were collected for RNA extraction.
2014-09-16 | E-GEOD-61416 | biostudies-arrayexpress
Project description:Samples were analyzed using a nanoACQUITY UPLC system (Waters) coupled to a Q-Exactive HF-X high resolution accurate mass tandem mass spectrometer (Thermo) via a nanoelectrospray ionization source. Briefly the sample was first trapped on a Symmetry C18 180 micron x 20 mm trapping column (5 microliters/min at 99.9/0.1 v/v H2O/MeCN) followed by an analytical separation using a 1.7 micron AQCUITY HSS T3 C18 75 micron x 250 mm column (Waters) with a 90 min gradient of 5 to 30% MeCN with 0.1% formic acid at a flow rate of 400 nl/min and column temperature of 55 degC. Data collection on the HF-X MS was performed in data-dependent acquisition (DDA) mode with a 120,000 resolution (at m/z 200) full MS scan from m/z 375 to 1600 with a target AGC value of 3e6 ions and 50 ms maximum injection time (IT). The Top30 peptides were selected for MS/MS using a resolution of 15,000, max AGC of 5e4 ions and minimum AGC target of 2.25e3, max IT of 45 ms, collision energy of 27, an isolation width of 1.2 m/z and 20 s dynamic exclusion.
2019-11-07 | MSV000084557 | MassIVE
Project description:Background: Prevention of hyperlipidemia and associated diseases is a health priority. Complementary medicine based on scientific evidence has recently recognized the potential of natural products for modulating lipid metabolism, such as the medicinal mushroom Ganoderma lucidum (Gl), which possesses hypocholesterolemic, prebiotic and antidiabetic properties. Methods: Whole-transcriptomic changes in liver and kidney from a mouse model (C57BL/6), under a high-cholesterol diet and standardized Gl extracts (Gl-1, Gl-2) or simvastatin administration, were analyzed to determine Gl hypocholesterolemic activity. Further effects of Gl extracts on lipid metabolism were evaluated using an in vitro hepatic-like macrophage model. Additionally, correlations among hepatic gene expression, microbiota and serum lipid profiles in vivo established by Gl extracts were evaluated. Results: Based on the hepatic and renal mRNA profiles of mice treated with Gl extracts and high-cholesterol diet, we identified relevant metabolic pathways modulated by Gl involving the restriction of lipid biosynthesis and the enrichment of lipid degradation and secretion. We further showed that Gl extracts induce a significant decrease of macrophage lipid storage and cholesterol biosynthesis, which occurs concomitantly by the down-modulation of Fasn and Elovl6. We also determined that prebiotic effects of Gl extracts modulating gut microbiota are correlated with the gene expression portraits. Conclusions: Our high-throughput analysis allowed to identify key transcriptomic nodes established by Gl extracts and their interaction with microbiome composition related to lipid catabolic signaling. Our results indicated that our Gl extracts have a robust potential to be used as transcriptome modulators and prebiotic agents to prevent metabolic disorders associated to hypercholesterolemia.
2020-12-15 | GSE159656 | GEO
Project description:M6A immunoprecipitation from 15 min BrU-labeled nascent RNA (0 min and 30 min chase). All three fractions, Input, eluate and supernatant were subjected to Illumina sequencing.
2018-06-19 | GSE114554 | GEO
Project description:Cells were solubilized with 8M urea followed by reduction with DTT and alkylation with iodoacetamide and overnight digestion with trypsin. After solid phase extraction, samples were labeled with TMT reagents as shown in metadata. Two pooled samples per plate were used for bridging two TMT sets. Reactions were quenched and samples mixed and desalted using high pH SPE. One ug of each TMT set was analyzed twice using a Waters nanoACQUITY interfaced to ThermoFisher Fusion Lumos. Trapping used a Symmetry C18 20 mm x 180 um trapping column (5 ul/min at 99.9/0.1 v/v water/acetonitrile), after which the analytical separation was performed using a 1.8 um Acquity HSS T3 C18 75 um x 250 mm column (Waters) with a 90 min linear gradient of 5 to 30% MeCN with 0.1% FA at a flow rate of 400 nL/min with a column temperature of 55C. Data collection Data-dependent acquisition used a r=120,000 (at m/z 200) full MS scan from m/z 375 - 1600 with a target AGC value of 2e5 ions. MS/MS scans were acquired at r=50,000 (at m/z 200) at a target AGC value of 1e5 ions and 105 ms. Precursor ions with a charge state of 2 and charge states of 3-6 had isolation windows of 1.2 and 0.7, respectively. A 20s dynamic exclusion was employed.
2024-07-10 | MSV000095293 | MassIVE
Project description:<p>The sample to be tested was fully ground into powder in the grinder, 50 mg of the sample was freeze-dried, 1000 μL of the extraction solution containing the inner target was added (methanol:acetonitrile:water, 2:2:1, v/v/v, internal standard concentration 20 mg/L) and vortex mixed for 30 s; then add the steel ball to the 45 Hz grinder for 10 min, ultrasonic 10 min; the sample to be tested is obtained after filtration. When detecting metabolites, metabolite determination was performed based on the LC-MS system, which mainly consists of Waters Acquity I-Class PLUS ultra-high performance liquid tandem and Waters Xevo G2-XS QTof high-resolution mass spectrometer. Meanwhile, the Waters Acquity UPLC HSS T3 column (1.8 um 2.1 x 100 mm) was used as the chromatographic column. Positive and negative ion modes were used to determine the metabolites. Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: 0.1% acetonitrile formate. The mobile phase conditions of liquid chromatography were as follows: the flow rate was 400 μL/min, 0.0 min: 98% flow A, 2% flow B; 0.25 min: 98% flow A, 2% flow B, 10.0 min: 2% flow A, 98% flow B; 13.0 min: 2% flow A, 98% flow B; 13.1 min: 98% flow A, 2% flow B; 15.0 min: 98% flow A, 2% flow B. MSe mode controlled by acquisition software (MassLynx v4.2, Waters) was used for primary and secondary mass spectrum data acquisition. ESI ion source parameters are as follows: Capillary voltage, 2500 V (positive ion mode) or -2000 V (negative ion mode); Cone hole voltage, 30 V; Ion source temperature, 100 °C; Desolvent temperature, 500 °C; Air flow rate, 50 L/h; Desolvent gas flow rate, 800 L/h; Plastic-nucleus ratio (m/z) collection range 50-1200 m/z. In the qualitative and quantitative analysis of metabolites, the original data collected by MassLynx v4.2 were processed by the Progenesis QI software for peak extraction, peak alignment, and other data, and the metabolites were identified based on the Progenesis QI software online METLIN database. Then, based on the results of the total score, MS2 score, and mass error (ppm), the metabolites were qualitatively determined.</p>
2024-07-12 | MTBLS10002 | MetaboLights
Project description:The goal of this study is to compare the transcriptome stability of DN1wt versus DN1gl/gl and DN1CD2-Ostm1gl/glTR Methods: DN1 thymocytes mRNA profiles of 19-day-old wild-type (WT), osteopetrotic grey-lethal (gl/gl) and transgenic CD2-Ostm1 gl/gl mice were generated by deep sequencing,DN1 cells from 2-3 mice per genotype were pooled, no technical replicates, using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: TopHat and Cufflinks. qRT–PCR validation was performed using SYBR Green assays Results: DN1 gl/gl showed a distinctive transcriptome signature in comparison to DN1 wt and DN1 gl/glTR with respectivelly 146 and 205 differentially expressed genes and 205. Migration genes were significantly affected in DN1 gl/gl samples and RAC1 and S1PR1 gene expressions were confirmed using RT-qPCR. Conclusions: RNA seq allowed us to identify the enhanced expression of migration genes (RAC1 and S1PR1) in DN1gl/gl, that were both normalized in the DN1 cells from transgenic gl/glTR mice, which suggests defective T cell migration associated to the osteopetrotic thymus phenotype.
2016-09-01 | GSE72184 | GEO