Dataset Information

6 Ocotea sp. dataset analyzed in DIA mode (ESI positive and negative)

ABSTRACT: 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.

INSTRUMENT(S): Xevo G2 Q-Tof

ORGANISM(S): Ocotea Lancifolia (ncbitaxon:881603) Ocotea Guianensis (ncbitaxon:128661) Ocotea Odorifera (ncbitaxon:128668) Ocotea Diospyrifolia (ncbitaxon:881600) Ocotea Porosa (ncbitaxon:1365881) Ocotea Notata

SUBMITTER: Daniela Aparecida Chagas-Paula

PROVIDER: MSV000094030 | MassIVE | Wed Feb 07 08:34:00 GMT 2024

REPOSITORIES: MassIVE

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LC-MS/DIA-based strategy for comprehensive flavonoid profiling: an Ocotea spp. applicability case.

Alves Matheus Fernandes MF Katchborian-Neto Albert A Bueno Paula Carolina Pires PCP Carnevale-Neto Fausto F Casoti Rosana R Ferreira Miller Santos MS Murgu Michael M de Paula Ana Claudia Chagas ACC Dias Danielle Ferreira DF Soares Marisi Gomes MG Chagas-Paula Daniela Aparecida DA

RSC advances 20240301 15

We introduce a liquid chromatography - mass spectrometry with data-independent acquisition (LC-MS/DIA)-based strategy, specifically tailored to achieve comprehensive and reliable glycosylated flavonoid profiling. This approach facilitates in-depth and simultaneous exploration of all detected precursors and fragments during data processing, employing the widely-used open-source MZmine 3 software. It was applied to a dataset of six Ocotea plant species. This framework suggested 49 flavonoid ...[more]

PMID: 38567345

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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:Samples for metabolomic analysis were prepared as follows. A 200 uL aliquot of bacterial cultures or control medium (prepared, as described in the above section) was mixed with 800 uL of 99.5 percent ethanol (Fisher Scientific) and subjected to 3 rounds of freeze thaw cycles: 10 minutes at -80C, 8 minutes at room temperature. Next, samples were spun down for 10 minutes at 16,000g, 4C to remove cell debris, and supernatants were transferred to new tubes. Ethanol supernatants were stored at -80C until further extraction. Each ethanol extraction was performed in two technical duplicates. The solid phase extraction (SPE) of the ethanol extracts was performed with the Oasis HLB 96-well plate (30 mg Sorbent per Well, 30 um Particle Size) (Waters Corporation) set up with a vacuum pump manifold. Each well of the plate was washed with 700 uL of 100 percent HPLC-grade methanol and equilibrated with 700 uL of HPLC-grade water. Next, 400 uL of ethanol extracts were added to the wells and allowed to slowly elute. Wells were next washed with 800 uL of 5 percent methanol in water. Metabolites from bacterial cultures or control media were eluted with 200 uL of 100 percent methanol. Vacuum up to 20 psi was applied for the wells that did not elute within one hour. The collected eluted metabolites were stored at -20 until the HPLC-MS analysis. The HPLC-MS analysis of extracted metabolites was performed with the use of a Dionex UltiMate 3000 UHPLC system (Fisher Scientific) coupled to a Bruker impact HD quadrupole time-of-flight mass spectrometer (Bruker). The chromatographic separation was performed on a Kinetex C18 1.7 um, 100A UHPLC column (50 mm x 2.1 mm) (Phenomenex), held at 40C during analysis. 5 uL of each sample was injected. Mobile phase A was water, 0.1 percent v:v formic acid, and mobile phase B was acetonitrile, 0.1 percent v:v formic acid. The solvent gradient table was set as follows: 2 percent B, increased to 10 percent B over 0.2 min, then to 100 percent B at 12 min, held at 100 percent B for 1.5 min, decreased back to 2 percent B in 0.5 min, followed by washout cycle and equilibration; total analysis time of 15 min. The scanned m/z range was 80-2000 Th; capillary voltage 4,500 V; nebulizer gas pressure 2 bar, drying gas flow rate 9 l min-1 and temperature 200C. The full MS scan was followed by a tandem mass spectrometry fragmentation of the seven most abundant ions within the spectrum. For tandem mass spectrometry, the collision cell collision energy was set at 3 eV and the collision energy was stepped 50 percent, 75 percent, 150 percent, and 200 percent. The scan rate was 3 Hz. A HP-921 lock mass compound was infused during the analysis for post processing mass correction.

Project description:Proteomic analysis Epilepsy was induced using perforant pathway stimulation (PPS) in rats as described (Norwood BA, et al. (2010) Classic hippocampal sclerosis and hippocampal-onset epilepsy produced by a single "cryptic" episode of focal hippocampal excitation in awake rats. J Comp 803 Neurol 518(16):3381-3407). Rats were killed under deep anesthesia (xylazine+ketamine) by transcardial perfusion with ice-cold 0.9 % NaCl solution at the following time points: 1 h, 24 h, 72 h, 10 d and 16 d after PPS (epileptogenesis); within 1 d of first spontaneous seizure (early epilepsy); 1 month after first spontaneous seizure (chronic epilepsy). Control rats were killed on day 17 after surgery (corresponding to day 10 after PPS). Hippocampi were removed and snap frozen at -80°C. Frozen rat hippocampi were resuspended in 200 ul lysis buffer (6M GdmCl, 10mM TCEP, Complete protease inhibitor cocktail, PhosSTOP inhibitor, 100 mM TEAB, benzonase) and dounced 30 times on ice. Samples were boiled for 5 min, sonicated on ice for 2min and centrifuged at 13000 rpm for 3 min. The supernatant was added chloroacetamide to a final concentration of 20 mM followed by incubation in the dark for 30 min at room temperature (RT). Proteins were digested with LysC for 30 min at RT and then diluting 10x— using 100 mM TEAB followed by adding trypsin and incubating overnight at 37°C with shaking. LysC and trypsin were added in a LysC/trypsin:protein ratio of 1:100. Samples were centrifuged at 13000 rpm for 5 min and from the supernatant a volume corresponding to 50 ug peptide was transferred to a new eppendorff tube. Sets of 6 samples were labelled using the TMTsixplex isobaric label reagent. (ThermoFisher Scientific). The labeling reagent was prepared using the manufactures instructions to first equilibrate to RT, then dissolving the labeling reagent in 41 ul anhydrous acetonitrile. The labeling reagent was added to each sample and incubated for 1 hour at RT. The reaction was quenched by incubating with 0.76 M lysine for 15 min. A small amount was taken from each sample, mixed and tested by mass spectrometry for labeling efficiency and mixing ratio. Remaining samples were mixed in a 1:1 ratio. The Stage-tips for the high-pH reverse phase fractionation were prepared by placing first two C18 disks in a p200 pipette tip, then adding a slurry of C18 beads (ReproSil-Pur, 3 um) in methanol and adding one additional C18 disk in the top. The column was activated and equilibrated by washing one time in 150 ul buffer B (50% buffer A, 50% ACN) and then two times in 150 ul buffer A (20mM Ammonium hydroxide, pH 10). The labeled peptide sample was adjusted to pH 10 using buffer A and then loaded onto the activated and equilibrated stage-tip by spinning the sample through the column and collecting 26 the flow-through. The column was washed 2 times with 150 ul buffer 585 A and the washes were collected and combined with the flow-through. Next, peptides were eluted stepwise in 17 fractions by spinning 150 ul of buffer A containing increasing amounts of ACN for each step (3.5%, 4.5%, 6%, 8%, 10%, 10.5%, 11.5%, 13.5%, 15%, 16%, 17.5%, 18.5%, 19.5%, 21%, 27%, 50%, 80%) through the column. After collection, the liquid was evaporated completely in a Concentrator Plus (Eppendorf) and prepared for MS by resuspending in buffer A* (2% ACN, 0.1% TFA). Samples were analyzed using an Easy-nLC system coupled online to a Q Exactive HF mass spectrometer (Thermo Scientific) equipped with a nanoelectrospray ion source (Proxeon/Thermo Scientific). The peptides were eluted into the mass spectrometer during a chromatographic separation from a fused silica column packed in-house with 3 um C18 beads (Reprosil, Dr. Maisch) using a 120 min gradient of buffer B (80% ACN, 0.5% acetic acid) with a flow rate of 250 nL/min. The Q Exactive HF was operated in positive ion mode with a top 12 data-dependent acquisition method where ions were fragmented by higher-energy collisional dissociation (HCD) using a normalized collisional energy (NCE)/ stepped NCE of 28 and 32. The resolution was set to 60,000 (at 400m/z), with a scan range of 300-1700 m/z and an AGC target of 3e6 for the MS survey. MS/MS was performed at a scan range of 100 - 2000 m/z using a resolution of 30,000 (at 400 m/z), an AGC target of 1e5, an intensity threshold of 1e5 and an isolation window of 1.2 m/z. Further parameters included an exclusion time of 45 sec and a maximum injection time for survey and MS/MS of 15 ms and 45 ms respectively.

Project description:Protein samples were measured using the LC-MS/MS platform containing reversed-phase nano liquid chromatography (nano Acquity M-class UPLC) (Waters Corporation, Massachusetts, USA), which was coupled to nanospray ionization tandem mass spectrometry with traveling wave ion mobility using high-definition data-independent (HD-MSE) acquisition and enabled with hybrid quadrupole orthogonal acceleration time of flight mass spectrometer (Synapt G2Si, Waters Corporation). The peptide mixture was separated on ACQUITY UPLC M-Class HSS T3 1.8 um, 75 um x 200 mm column (Waters Corporation) using a mixture of Buffers A (0.1% v/v Acetic acid in water) and B (0.1% v/v acetic acid in acetonitrile) through a linear concentration gradient of Buffer B in 170 min, with a flow rate of 300 nl/min from 5-26% (v/v). The eluents were sprayed at a voltage of 1.85-1.90 kV using PicoTip emitters (Waters Corporation), and other source parameters were set (sampling cone = 40V, source offset = 0 V, source temperature = 80C, gas flows, cone gas = 50 l/h, nano gas flow = 0.4 bar, purge gas = unchanged, IMS = optimized for wave velocity with a start velocity of 870 m/s to end at 564 m/s that corresponds to the separation of GluFib fragments in the drift time range of 0-200 bins). The data was acquired using the MassLynx software program version V4.1 (Waters Corporation) that automatically switches between MS and MS/MS (HDMSE) scans in resolution mode at 20000 with 1 second (sec) scan time and the scan range of 50-2000 m/z. Calibration was done by injecting GluFib at an interval of 1 min. The acquired data were analyzed for protein identifications against the Uniprot fasta database of B. cereus ATCC14579 strain that contained 5240 sequences via the PLGS v3.3 program (Water Corporation). Spectra were processed using low and high-energy thresholds of 135, 20 counts, and lock mass calibration 785.8456 m/z for GluFib. The workflow search parameters were set as: (protease = trypsin, one missed cleavage, carbamidomethyl for cysteine as fixed, variable modifier = oxidation of methionine). The protein was quantified when 1) the top 3 peptides had no modifications; 2) pass one match had peptide fragment one; and 3) ranked the first three highest peptides. The PLGS independent identification output was imported to ISOQuant 1.8 (Distler et al., 2014) for sample analyses. [doi:10.25345/C5Z892K4R] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Dataset Information

6 Ocotea sp. dataset analyzed in DIA mode (ESI positive and negative)

Dataset's files

Publications

LC-MS/DIA-based strategy for comprehensive flavonoid profiling: an <i>Ocotea</i> spp. applicability case.

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