Dataset Information

Beta-cell Galphas signaling is critical for physiological and pharmacological enhancement of insulin secretion

ABSTRACT: Islet lysates were adjusted to 5 percent SDS in TEAB, pH 8.5, reduced and alkylated and digested with modified trypsin (Worthington) using an S-trap mini device (Protifi). Peptides were labeled with TMT16 reagents (lot ZH350094). Phosphopeptide enrichment used GL Sciences p10 TiO tips. LC-MS/MS used a MClass UPLC system (Waters) and Exploris 480 (ThermoFisher). Analytical separation used HSS T3 C18 75 micron by 250 mm column (Waters) with a 90 min gradient of 5 to 30 percent MeCN at a flow rate of 400 nl per min. Data collection on TMT-labeled samples was performed in data-dependent acquisition (DDA) mode with 2 FAIMS compensation voltages. TMT data was analyzed using Fragpipe 19.

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

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Publications

Branched-chain α-ketoacids are preferentially reaminated and activate protein synthesis in the heart.

Walejko Jacquelyn M JM Christopher Bridgette A BA Crown Scott B SB Zhang Guo-Fang GF Pickar-Oliver Adrian A Yoneshiro Takeshi T Foster Matthew W MW Page Stephani S van Vliet Stephan S Ilkayeva Olga O Muehlbauer Michael J MJ Carson Matthew W MW Brozinick Joseph T JT Hammond Craig D CD Gimeno Ruth E RE Moseley M Arthur MA Kajimura Shingo S Gersbach Charles A CA Newgard Christopher B CB White Phillip J PJ McGarrah Robert W RW

Nature communications 20210315 1

Branched-chain amino acids (BCAA) and their cognate α-ketoacids (BCKA) are elevated in an array of cardiometabolic diseases. Here we demonstrate that the major metabolic fate of uniformly-13C-labeled α-ketoisovalerate ([U-13C]KIV) in the heart is reamination to valine. Activation of cardiac branched-chain α-ketoacid dehydrogenase (BCKDH) by treatment with the BCKDH kinase inhibitor, BT2, does not impede the strong flux of [U-13C]KIV to valine. Sequestration of BC ...[more]

PMID: 33723250

Similar Datasets

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.

Project description: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.

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.