MS and tandem MS data were collected on the Thermo Scientific Orbitrap Exactive Plus mass spectrometer modified and optimized for transmission and detection of ions with m/z up to 50 kTh as described previously [37 (link)]. For ion isolation, we used a standard quadrupole mass filter from a Q-Exactive instrument with a modified electronic board featuring a decreased resonance frequency of 284 kHz enabling an upper mass-selection limit above 20 kTh [38 (link), 39 (link)]. The samples were buffer exchanged into 10 mM ammonium acetate at pH 6.8 and diluted to a final concentration of 10 μM immediately before the experiment. All acquisitions were collected at the 64-ms transient times equivalent to 17,500 resolution at m/z 200. For each final spectrum, a minimum of 10 scans was combined, containing 10 μscans at 100 ms injection times. In all MS and IM-MS experiments, the samples were sprayed from gold-coated borosilicate glass needles produced in-house [40 (link)].
Exactive plus orbitrap
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States
The Exactive Plus Orbitrap is a high-resolution mass spectrometer designed for accurate mass measurement and identification of molecular compounds. It utilizes Orbitrap technology to provide high mass resolving power and mass accuracy. The core function of the Exactive Plus Orbitrap is to precisely analyze the mass-to-charge ratio of ions, enabling the identification and characterization of a wide range of molecules.
Automatically generated - may contain errors
Lab products found in correlation
Sequant zic philic, by Merck Group (10 mentions)
Sequant guard column, by Merck Group (10 mentions)
Acquity 1 class uplc system, by Thermo Fisher Scientific (3 mentions)
Ultimate 3000, by Thermo Fisher Scientific (3 mentions)
Tracefinder, by Thermo Fisher Scientific (2 mentions)
I class uplc system, by Thermo Fisher Scientific (2 mentions)
Uplc system, by Thermo Fisher Scientific (2 mentions)
Xcalibur v 4, by Thermo Fisher Scientific (2 mentions)
Easy nlc 1000 uplc system, by Thermo Fisher Scientific (1 mentions)
Acclaim pepmap100, by Thermo Fisher Scientific (1 mentions)
C18 ziptip, by Merck Group (1 mentions)
Acclaim pepmap 100 c18, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000lc, by Thermo Fisher Scientific (1 mentions)
Bio bond c4 column, by Dikma Technologies (1 mentions)
Nexus 670 ft ir spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Am 400 spectrometer, by Bruker (1 mentions)
Zorbax sb c18 column, by Agilent Technologies (1 mentions)
Q exactive plus quadrupole orbitrap, by Thermo Fisher Scientific (1 mentions)
Avance 400 nmr spectrometer, by Bruker (1 mentions)
Agilent 1200, by Agilent Technologies (1 mentions)
54 protocols using exactive plus orbitrap
1
Optimized Ion Detection and Isolation
2
Peptide Analysis by Mass Spectrometry
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A mass spectrometer (Thermo Scientific Q
Exactive Plus) was used to analyze the enriched peptides, which were
cleaned using C18 Zip Tips (Millipore, Bedford, MA, USA). The peptides
were loaded onto a column (Acclaim PepMap 100, 100 μm ×
2 cm, nanoViper C18, Thermo Fisher Scientific Inc., Waltham, MA, USA)
connected to a reversed-phase analytical column (Acclaim PepMap 100
C18, 75 μm, 150 mm, 3 μm, Thermo Fisher Scientific Inc.,
Waltham, MA, USA) in 0.1% formic acid (solvent A) and then isolated
with a linear gradient of 0.1% formic acid and 90% acetonitrile (solvent
B) at a flow rate of 350 nL/min on an EASY-nLC 1000 UPLC system (Thermo
Fisher Scientific Inc). The gradient was as follows: 0–60 min,
6–24% solvent B; 60–82 min, 24–36% solvent B;
82–86 min, 36–80% solvent B; and 86–90 min, solvent
B at 80%. The peptides were analyzed using MS/MS in an Orbitrap Exactive
Plus (Thermo Fisher Scientific Inc) coupled with an online UPLC system.
An electrospray voltage of 2.0 kV was applied. To read MS scans, the m/z scan range was set at 350 to 1550. The intact peptides
were detected at a resolution of 60,000, whereas the resolution for
higher-energy collisional dissociation (HCD) spectra was 17,500. The
automatic gain control (AGC) target was set to 1E4, and the maximum
injection time was 100 ms with a 15 s dynamic exclusion duration.
Exactive Plus) was used to analyze the enriched peptides, which were
cleaned using C18 Zip Tips (Millipore, Bedford, MA, USA). The peptides
were loaded onto a column (Acclaim PepMap 100, 100 μm ×
2 cm, nanoViper C18, Thermo Fisher Scientific Inc., Waltham, MA, USA)
connected to a reversed-phase analytical column (Acclaim PepMap 100
C18, 75 μm, 150 mm, 3 μm, Thermo Fisher Scientific Inc.,
Waltham, MA, USA) in 0.1% formic acid (solvent A) and then isolated
with a linear gradient of 0.1% formic acid and 90% acetonitrile (solvent
B) at a flow rate of 350 nL/min on an EASY-nLC 1000 UPLC system (Thermo
Fisher Scientific Inc). The gradient was as follows: 0–60 min,
6–24% solvent B; 60–82 min, 24–36% solvent B;
82–86 min, 36–80% solvent B; and 86–90 min, solvent
B at 80%. The peptides were analyzed using MS/MS in an Orbitrap Exactive
Plus (Thermo Fisher Scientific Inc) coupled with an online UPLC system.
An electrospray voltage of 2.0 kV was applied. To read MS scans, the m/z scan range was set at 350 to 1550. The intact peptides
were detected at a resolution of 60,000, whereas the resolution for
higher-energy collisional dissociation (HCD) spectra was 17,500. The
automatic gain control (AGC) target was set to 1E4, and the maximum
injection time was 100 ms with a 15 s dynamic exclusion duration.
3
Lipid Profiling by High-Resolution LC-MS
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Lipid samples were analyzed at the Harvard Center for Mass Spectrometry. The LC–MS analyses were modified from Miraldi81 (link) and were performed on an Orbitrap Exactive plus (Thermo Scientific) in line with an Ultimate 3000 LC (Thermo Scientific). Each sample was analyzed in positive and negative modes, in top 5 automatic data-dependent MSMS mode. Column hardware consisted of a Biobond C4 column (4.6 × 50 mm, 5 μm, Dikma Technologies). Flow rate was set 100 μl min−1 for 5 min with 0% mobile phase B (MB), then switch to 400 μl min−1 for 50 min, with a linear gradient of MB from 20 to 100%. The column was then washed at 500 μl min−1 for 8 min at 100% MB before being reequilibrated for 7 min at 0% MB and 500 μl min−1. For positive mode runs, buffers consisted for mobile phase A (MA) of 5 mM ammonium formate, 0.1% formic acid and 5% methanol in water, and for mobile phase B (MB) of 5 mM ammonium formate, 0.1% formic acid, 5% water, 35% methanol in isopropanol. For negative runs, buffers consisted for MA of 0.03% ammonium hydroxide, 5% methanol in water, and for MB of 0.03% ammonium hydroxide, 5% water, 35% methanol in isopropanol. Lipids were identified and quantified using the Lipidsearch© software (version 4.2.27, Mitsui Knowledge Industry, University of Tokyo). Integrations and peak quality were curated manually before exporting and analyzing the data in Microsoft Excel.
4
Metabolomic Profiling of Malaria Parasite
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Changes in metabolites were measured in response to compounds using whole cell hydrophilic extraction, followed by ultra-high precision liquid chromatography mass-spectrometry (UHPLC-MS) using negative ionization as in Cowell et al., 2018 (Cowell et al., 2018 (link)). This was performed on synchronous, trophozoite infected red blood cells (iRBCs, 24–36 hpi) which had been magnetically separated from culture. Quantification of cells was performed by hemocytometry, and treatments were performed on 1 × 108 iRBCs in wells containing 5 mL of RPMI. Treatment conditions were performed in triplicate, with compound concentrations of 10xEC50 for 2.5 hr, followed by washing with PBS and extraction using 90% methanol containing isotopically-labeled aspartate as an internal standard for sample volume. Samples were dried using nitrogen prior to resuspension in water containing 0.5 uM chlorpropamide as an internal standard for injection volume. Samples were then analyzed via UHPLC-MS on a Thermo Scientific EXACTIVE PLUS Orbitrap instrument as established in Allman et al., 2016 (Allman et al., 2016 (link)).
5
Detailed Synthesis and Characterization
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Reagents for synthesis were obtained from Sigma-Aldrich (St. Louis, MO, USA). 1H and 13C-NMR spectra (400 MHz for proton and 100 MHz for carbon) were recorded on an AM-400 spectrometer (Bruker, Rheinstetten, Germany); IR spectra (KBr pellets, 500–4000 cm−1) were recorded on a NEXUS 670 FT-IR spectrophotometer (Thermo Nicolet, Madison, WI, USA). Mass spectrometry was conducted in a High resolution mass spectrometer Exactive™ Plus Orbitrap (ThermoFisher Scientific, Bremen, Germany), Scan parameters: Resolution: 140,000, AGC target: 3e6, Max. inject time: 200, HESI source: Sheath gas flow: 10, Aux gas flow rate: 3, Sweep gas flow rate: 0, Capillary temp.: 250 °C, S-lens RF level: 0, Heater temp: 50 °C.
6
Metabolic Profiling of LGG Planktonic Cultures
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LGG planktonic cultures grown in 50% TSB, TSB supplemented with (1% w/v) D-(+) - glucose, (1% w/v) D-(+)- raffinose or (1% w/v) D- (+)- mannose for 24 h at 37 °C. To get an equal number of bacteria the OD600 of the bacteria was compared, and after that washed twice with PBS. For polar metabolite analysis in the polar phase samples, the lyophilized pellets were dissolved using 100 µL DDW-methanol (1:1), centrifuged twice (at maximum speed) to remove possible precipitants, and were injected into LC-MS system. Polar analysis in the polar phase was done as following: Analysis was performed using Waters Acquity I class UPLC System combined with a mass spectrometer (Thermo Exactive Plus Orbitrap) operated in a negative ionization mode. The LC separation was done using the SeQuant Zic-pHilic (150 mm × 2.1 mm) with the SeQuant guard column (20 mm × 2.1 mm) (Merck). The Mobile phase B: acetonitrile and Mobile phase A: 20 mM ammonium carbonate with 0.1% ammonia hydroxide in water: acetonitrile (80:20, v/v). The flow rate was kept at 200 μL min−1 and gradient as follows: 0–2 min 75% of B, 17 min 12.5% of B, 17.1 min 25% of B, 19 min 25% of B, 19.1 min 75% of B, 23 min 75% of B.
7
Targeted Metabolomics Analysis by HPLC-Orbitrap MS
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Labelled samples and mock samples were measured with an UltiMate 3000 HPLC system coupled to an Exactive Plus Orbitrap mass spectrometer (both from Thermo Fisher Scientific). Ten microlitres of sample solution was injected into the system. For chromatographic separation, a Zorbax SB-C18 column (150 × 2.1 mm, 3.5 μm; Agilent Technologies) was used at 25 °C and at a flow rate of 250 μL min−1. Mobile phases consisted of H2O (eluent A) and MeOH (eluent B), both containing 0.1 % FA (v/v) and 5 mM NH4HCO2. Gradient method 1 was as follows: 0–0.5 min, 10 % B; 0.5–20.0 min, 10–100 % B; 20.0–25.0 min, 100 % B; 25.0–25.1 min, 100–10 % B; and 25.1–30.0 min, 10 % B. Mass spectrometric analysis was performed in fast polarity switching mode using electrospray ionisation. Applied settings were similar to Kluger et al. [16 (link)] with some modifications: Automatic gain control was set to 5 × 105, and a maximum injection time of 500 ms was used. Full scan measurement was carried out in the scan range of m/z 130–1,300 with a resolution of 70,000 full width at half maximum (FWHM) at m/z 200. The instrument was calibrated with Pierce Ion Calibration Solution in both polarity modes prior to analysis. For data evaluation, software Thermo Xcalibur 2.2 was applied.
8
High-Resolution Mass Spectrometry Analysis
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High-resolution mass spectrometry detection was performed on both Exactive Plus Orbitrap and Q-Exactive Plus Quadrupole-Orbitrap mass spectrometers (Thermo) equipped with an electrospray ionization source and operated in positive mode. Screening and confirmation of the substances were performed using full scan mode in Exactive Plus and parallel reaction monitoring (PRM) mode in Q-Exactive Plus. For identification of selected molecules, the scan range and collision energy were applied to each substance at a resolution of 35,000 in PRM mode.
9
Targeted Metabolomic Profiling and Analysis
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Following negative ionization analysis of hydrophilic extracts on a Thermo Exactive Plus Orbitrap, sample data were converted and transferred for analysis. Targeted peak picking from a curated list of 298 metabolites was achieved using el-MAVEN software (https://elucidatainc.github.io/ElMaven/ (Agrawal et al., 2019 (link))), followed by normalization and analysis via RStudio (http://www.rstudio.com/ ) and Metaboanalyst (https://www.metaboanalyst.ca/ (Chong et al., 2018 (link))). Data were visualized using the Hyperspec (http://hyperspec.r-forge.r-project.org ) and Suprahex R (Fang and Gough, 2014 ) scripting packages in RStudio. Hierarchical clustering of the metabolic profiles to identify related metabolic signatures was performed using the Ward method, based on the Pearson correlation coefficients, by the Hyperspec R integrated heatmap function.
10
Antibody Characterization by LC-ESI-MS
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