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>

Project description:This dataset consists of 6 Ocotea plant species analyzed in negative and positive modes, with data-independent acquisition (MSe), in a Waters Xevo G2 instrument. The available data were converted in .mzML format using the Waters2mzML software, available on Github. Detailed information about data acquisition: For MSe, data acquisition was performed in a UPLC system coupled to an ESI source and a QTOF XevoTM G2 instrument (Waters Corp., Milford, MA, USA). Chromatographic separation was achieved using a C18 column (100 x 2.1 mm and 1.8 microm particle diameter, ACQUITY UPLC HSS T3), and a mobile phase gradient with a flow rate of 0.5 mL/min, composed of ACN/water, both phases acidified with 0.1% formic acid. The runtime was 10 minutes, with an injected sample volume of 5 microL, and the oven and shelf temperatures 40 C and 10 C, respectively. The chromatographic gradient began with an initial composition of 1 percent ACN and, was followed by a transition to 15 percent ACN at 0.1 min. Further changes in solvent composition occurred at 7.5 min (80 percent ACN), 8.5 min (99 percent ACN), and 8.6 min (1 percent ACN) until 10 min. The mass spectrometer was operated in MSe acquisition mode with alternating high and low-energy scans, for positive and negative ionization modes. The ionization energy was set at 3 eV for low-energy scans and 25-40 eV for high-energy scans. ESI-MS at a resolution up to 40.000 with a full mass scan range set from 50 to 1000 m/z for functions 1 and 2 was applied. Instrument parameters, including cone voltage (40 V), capillary voltage (3.0 kV), cone gas flow (30 L/h), auxiliary gas flow rate (10 L/h); desolvation temperature (300 C), source temperature (120 C), and desolvation gas flow (600 L/h), were carefully optimized. High-purity nitrogen was employed for desolvation, collision, and cone gas. To ensure accuracy and reproducibility, a solution of leucine-encephalin was used as a lock mass with m/z 554.2622 (ESI-) and m/z 556.2768 (ESI+) for identification. MS data were continuously collected, and lock spray calibration was performed every 10 seconds.

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: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:Methods 13C6 Lysine isotope labeling study. Male Bl6/N mice (12 weeks old) were fed a custom diet (Silantes) containing more than 99% 13C6 lysine. The protocol for labeling was described previously. After 1, 2, and 8 weeks (protocol 1) or 1, 2, and 3 weeks (protocol 2), mice were sacrificed and perfused with ice-cold PBS. All denominated organs were snap-frozen from the same mouse and stored at -80C degrees. Blood was separated in erythrocyte and plasma by centrifugation. Metabolomics sample preparation. Frozen organs were kept on dry ice and 10 mg tissue was weighed. Then, 800 ul ice-cold extraction solution containing Acetonitril : Methanol : Water 2:2:1 was added. Samples were subjected to tissue homogenization using a multiplex-bead-beater (Storm) for 30 sec (liver), 1 min (lung), or 45 sec (all other tissues). The supernatant was transferred to a prechilled Eppendorf tube. Then, beads were washed with 200 ul of the extraction solution, and the wash solution was added to the remaining homogenate. The homogenate was incubated at -20C degrees for 2 hrs. Then, tissue was spun down at 4 C degrees for 20 min, and the supernatant was transferred to another vial. The supernatant containing the extract was transferred into a speed vac and dried down. The next morning, the dried-down extract was once resuspended in 100 ul (per 10 mg tissue) of acetonitrile-water 1:1, and the solution was centrifuged at 4 C degrees. Then, the solution was transferred to autosampler vials and stored at -80 C until further use. Untargeted metabolomics analysis and mass spectrometry. LC-MS/MS analysis for metabolomics was performed as previously described. Data was annotated with in-source fragments and adducts. The mass spectrometer was initially calibrated using NaFormate peaks and in addition post-run. For untargeted metabolomic analysis, we used a UHPLC-MS approach. For fractionation, we used hydrophilic interaction liquid chromatography (HILIC) fractionation and reversed-phase (RP) chromatography as previously described22. We used a quadrupole time-of-flight instrument (Impact II, Bruker, Bremen, Germany) coupled to a ultrahigh-performance liquid chromatography (UHPLC) device (Bruker Elute, Bruker, Billerica, MA), or to an Agilent Infinity 1290 UHPLC device (Agilent, USA). The MS was calibrated using sodium formate (post-run mass calibration). Data were acquired over an m/z range of 50 to 1000 Da in positive ion mode and negative ion mode (HILIC only). Electrospray source conditions were set as follows: end plate offset, 500 V; dry gas temperature, 200C; drying gas, 6 liters/min; nebulizer, 1.6 bar; and capillary voltage, 3500 V. To increase metabolome coverage and minimize ion suppression, we used a dual fractionation strategy. For RP separation, an ACQUITY BEH C18 column (1.0 x 100 mm, 1.7-um particle size; Waters Corporation, Milford, MA) was used, and for HILIC fractionation, a ACQUITY BEH amide (1.0 x 100 mm, 1.7-um particle size; Waters Corporation, Milford, MA) column was used. Flow was 150 ul/min, and a binary buffer system consisting of buffer A (0.1% FA) and buffer B (0.1% FA in acetonitrile) was used. The gradient for RP was: 99% A for 1 min, 1% A over 9 min, 35% A over 13 min, 60% A over 3 min, and held at 60% A for an additional 1 min. The gradient for HILIC consisted of 1% A for 1 min, 35% A over 13 min, 60% A over 3 min, and held at 60% A for an additional 1 min. The injection volume was always 2 ul. For molecule identification purposes, putative molecules of interest were fragmented using three different collision energies (10, 20 eV) or ramp collision energies (20 to 50 eV). Untargeted metabolomics data analysis. Bruker Raw files (*.d) were transformed into mzml files using the compassxport_converter.py script (Bruker). Then, data files were uploaded to XCMS online, and differential peaks were extracted in positive and negative ion mode50. Feature Detection was performed with the centwave method with the following options: ppm =10, minimum peak width = 5, maximum peak width = 20, mzdiff = 0.01, Signal/Noise = 6, Integration method =1, prefilter peaks =3, prefilter intensity = 100, noise filter = 100. For Retention time correction, we used obiwarp method with profStep = 1. Alignment was perfomed with bw 05, minfrac = 0.5, mzwid = 0.015. mminsamp =1, and max = 100. Statistical test was performed using an unpaired parametric t-test (Welsh), with post-hoc analysis = TRUE,. Statistical filtering was performed for priorization of features of interest, including a fold change of at least 1.5, and a p-value lower than 0.05, and a corrected p value (q-value) lower than 0.05. The analysis included PCA and multivariate analyses. Metabolites were identified based on 1) unique mass, 2) MS2 spectra comparison to authentic standard 3) coelution with authentic standard, and 4) isotopic pattern. The following adducts were routinely considered : Na, H, for positive mode, and Cl, formate for negative mode.

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Raw Data for Anthocyanin identification from mixed flavonoid populations in flowers by positive and negative mode LCMSMS

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