P. murinus lyophilized fractions were dissolved in 100 µL of extracellular solution supplemented with 0.1% BSA and tested using manual whole-cell patch clamp on HEK293 cells stably overexpressing CaV3.2 (see protocol above). Fraction 32 was re-purified by RP-HPLC (UltiMate® 3000 Standard LC systems, ThermoFisher Scientific) on a C18 column (Synchronis C18, 254 ⨯ 4.6mm, 5 µm, ThermoScientific) using a gradient of water/ACN in 0.1% formic acid from 20-40% ACN over 20 min. Fractions were collected manually. Sub-fraction 32.6 was evaluated for activity using manual whole-cell patch clamp on HEK293 cells stably overexpressing CaV3.2 (see protocol below) and analyzed by LC-HRMS (Acquity H-Class, Waters, Synapt G2-S, Waters) using a Kinetex C18 100 Å column (100 × 2.1 mm, 2.6 μm particle size) from Phenomenex (France) and a gradient of water/ACN in 0.1% formic acid from 20-40% ACN over 20 min. Peptide mass was determined using electrospray ionization time-of-flight (ESI-TOF) mass spectrometry (Acquity H-Class, Waters, Synapt G2-S, Waters), and the amino acid sequence of the peptide was determined by Edman degradation.
Synapt g2 s
Manufactured by Waters Corporation
Sourced in United Kingdom, United States
The Synapt G2-S is a high-resolution mass spectrometry system designed for advanced analytical applications. It features a quadrupole time-of-flight (QTOF) mass analyzer that provides accurate mass measurements and high-resolution capabilities. The system is capable of performing sensitive and reliable quantitative and qualitative analyses across a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Acquity uplc, by Waters Corporation (5 mentions)
Acquity uplc 1 class, by Waters Corporation (3 mentions)
Acquity uplc beh c18 column, by Waters Corporation (2 mentions)
Masslynx 4, by Waters Corporation (2 mentions)
Aviii300, by Bruker (1 mentions)
Aviii400, by Bruker (1 mentions)
Elix gradient a10 system, by Merck Group (1 mentions)
Xevo g2 xs, by Waters Corporation (1 mentions)
Daidzein, by Tokyo Chemical Industry (1 mentions)
Formononetin, by Tokyo Chemical Industry (1 mentions)
Udp α d glucose, by Merck Group (1 mentions)
Biochanin a, by Tokyo Chemical Industry (1 mentions)
Genistein, by Tokyo Chemical Industry (1 mentions)
Xbridge c18 column, by Waters Corporation (1 mentions)
Ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Acquity uplc beh amide column, by Waters Corporation (1 mentions)
Orbitrap elite hybrid ion trap orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Proteome discoverer, by Thermo Fisher Scientific (1 mentions)
Pepsin, by Merck Group (1 mentions)
Q tof premier, by Waters Corporation (1 mentions)
68 protocols using synapt g2 s
1
Purification and Characterization of CaV3.2 Modulator
2
Synthesis and Characterization of 5-Iodo-dUTP
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Reagents and solvents were purchased from commercial suppliers and used without further purification, unless otherwise stated. 5′-O-(4,4′-dimethoxytrityl)-5-iodo-2′-deoxyuridine 2 was synthesised following an established procedure.20 (link) Column chromatography was carried out using open columns packed with Merck grade 60 silica gel topped with 0.5 cm of sand. TLC analysis was performed on Merck silica gel 60 silica sheets. 1H, 13C, and 31P NMR spectra were obtained on Bruker AVIII300 or AVIII400 spectrometers. Chemical shifts (δ) are given in ppm and are relative to the residual solvent peak. Electrospray mass (ESI-MS) spectra were measured by either Waters micromass LCT electrospray time-of-flight (ES-TOF), Waters Xevo G2-XS, or Synapt G2S mass spectrometers. Milli-Q water purified with a Millipore Elix-Gradient A10 system (resistivity > 18 μΩ cm, TOC ≤ 5 ppb, Millipore, France) was used for DNA sample preparation.
3
MS Analysis of Amyloid Peptide Oligomers
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The conditions used for MS, using a Waters Synapt G2-S, were as follows: positive polarity in sensitivity mode; capillary = 2.5 kV; nebulizer = 2 mbar; source temperature = 80 °C; desolvation temperature = 60 °C; sample cone setting = 35 V; source offset setting = 60 V; and mass range = 500–4000 m/z. These conditions were maintained throughout the study to ensure consistency of the data and to avoid influencing the detection of oligomers due to preferential ionization conditions.
Samples were directly infused into the mass spectrometer at a flow rate of 10 µl/min using a Protea PM-1000 Syringe Pump and Hamilton 1-ml syringe. The data acquisition of the amyloid peptide was performed using a Waters Synapt G2-S quadrupole time of flight mass spectrometer (Q-TOF MS) with traveling wave ion mobility (Waters Corp., Milford, MA, USA). The data were acquired using the systems sensitivity mode to allow for the detection of the less abundant oligomers. Samples were infused at room temperature. The IMS–MS studies were conducted at Protea, Inc. (Morgantown, WV, USA).
Samples were directly infused into the mass spectrometer at a flow rate of 10 µl/min using a Protea PM-1000 Syringe Pump and Hamilton 1-ml syringe. The data acquisition of the amyloid peptide was performed using a Waters Synapt G2-S quadrupole time of flight mass spectrometer (Q-TOF MS) with traveling wave ion mobility (Waters Corp., Milford, MA, USA). The data were acquired using the systems sensitivity mode to allow for the detection of the less abundant oligomers. Samples were infused at room temperature. The IMS–MS studies were conducted at Protea, Inc. (Morgantown, WV, USA).
4
Purification and Characterization of YjiC
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Genistein, daidzein, biochanin A and formononetin were purchased from Tokyo Chemical Industry (Japan). UDP-α-D-glucose was purchased from Sigma-Aldrich (USA). Genistein 7-O-β-D-glucoside (genistin) was available in our laboratory. All other chemicals and reagents were of the highest chemical grade available. The high resolution mass spectrometry spectra were obtained in positive ion mode on ACQUITY (UPLC, Waters Corp., USA) coupled with SYNAPT G2-S (Waters Corp.). The details of the methodology for the cloning, expression, and purification of YjiC are described in our previous report (Pandey et al., 2013a (link)).
5
LC-MS/MS Analysis of Ribosomal RNA
6
Ion Mobility Mass Spectrometry of Proteins
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Experiments were performed on a Waters Synapt G2S instrument using NanoESI and trap-activated ion mobility; capillary voltage was 1.2–1.5 kV, cone 40 V, and source temperature 40 °C. The helium cell and the IMS gas flows were 180 and 90 ml/min, respectively; the IMS wave velocity was 400 m/s, and the IMS wave height was 35 V. Nitrogen was the carrier gas. The most intense charge state for each protein species was mass selected using the quadrupole prior to the trap region. Activation was induced by elevating the trap collision energy. ORIGAMI was used to automatically acquire data for collision energies from 4 to 200 V in 2-V increments, as well as for data processing (39 (link)).
7
Real-time Breath Analysis using QTOF-MS
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The experimental set-up has been presented in detail previously [22] (link), [23] (link), [26] (link), [27] (link). Briefly, the entrance of a commercial quadrupole time-of-flight mass spectrometer (Synapt G2S, Waters, UK) was slightly modified to allow for the admission of breath samples through a heated Teflon tube. Exhaled breath was mixed with a nano-electrospray plume (water, 0.2% formic acid), whereby some compounds present in breath were readily detected in real time. The mass spectrometer was operated in the positive ion mode. The scans were recorded using the mass-lock option, using a common mass spectroscopy (MS) contaminant as lock mass (i.e., protonated phthalic anhydride, mass-to-charge-ratio (m/z) 149.0233 [28] (link)). The data analysis work-flow is given in Fig. 1 .
8
Chitinase Profiling of Tunicate Membranes
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Purified envelope membranes of C. intestinalis Type A, B. floridae, E. atami and O. mossambicus were separately treated with recombinant hyperthermophilic chitinase PF-ChiA from Pyrococcus furiosus (Thermostable Enzyme Laboratory, Japan) following manufacturer’s instructions. An aliquot (5 μL) was collected from each reaction at 0, 1, 2, 3 and 4 h after the onset of reaction and subjected to LC-MS analyses. For LC, we used an ACQUITY UPLC-I-class (Waters, USA) equipped with an ACQUITY UPLC BEH amide column and performed with a 5-min gradient from 80/20 to 25/75 MeCN/H2O with 0.1% NH4OH at a flow rate of 0.17 mL/min. For MS, we used a SYNAPT G2-S (Waters, USA) in ES+ ionization mode with capillary voltage at 2.0 kV, cone voltage at 25 V, source temperature at 120 °C desolvation temperatures at 350 °C, a desolvation gas flow of 500 L/h, cone gas flows of 50 L/h, scan time of 0.2 ms at a high resolution mode (35,000 full-width at half maximum) and a lock mass of Leu-enkephalin (556.27 m/z) at every 60 s.
9
Native MS Analysis of TraG*R100
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TraG*R100 samples were prepared for MS analysis using a 5 kDa molecular weight cutoff (MWCO) dialysis cassette to buffer exchange the protein at 4 °C into 100 mM ammonium acetate (MS grade), pH 6.6. Native MS analysis was performed on a Synapt G2S (Waters) with 5 μM TraG*R100 or ΔTraG*R100 flowing at a rate of 5 μl/min. In the collision induced unfolding (CIU)–MS experiments, data were collected from 5 V trap collision energy (CE) to 150 V in 5 V increments, with an acquired m/z range of 2000–5000 with no manual trapping. Capillary voltage was set to 3.0 kV, the sampling cone was 150.0, with source and desolvation temperatures at 120 and 250 °C, respectively, cone gas flow 71.0 l/h, nanoflow gas pressure at 2 bar, desolvation gas flow at 600 l/h, the transfer CE was 10.0 V, the trap gas flow was 4.0 ml/min, and the ion mobility spectrometry (IMS) wave delay was 1 ms with a wave height start of 10 V and a wave end height of 40 V.
10
Rapid Protein Digestion and Analysis
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The CFI-TRESI-HDX experiments
were completed according to the experimental setup shown inFigure 1 . Construction of
the capillary-based sub-second mixing device has been previously described.29 (link),30 (link) PerkinElmer Series 200 pumps and autosamplers were used to deliver
solvents. Pepsin (porcine gastric mucosa, Sigma-Aldrich) or Protease
XIII (Aspergillus saitoi, Sigma-Aldrich) was cross-linked in-house
onto NHS-activated agarose (PierceTM, Thermo Fisher Scientific). Digestion
columns were constructed in-house using PEEK tubing with an ID of
0.040” and a 2 μm pore-size frit upstream of the ESI
emitter. All data were acquired on a Waters Synapt G2-S, and IMS was
employed in the TriWave cell to improve the spatial resolution of
peptides in the digested samples. Peptide identification was performed
using Proteome Discoverer (Thermo Fisher Scientific) after LC-MS/MS
analysis with an Orbitrap Elite Hybrid Ion Trap-Orbitrap Mass Spectrometer.
were completed according to the experimental setup shown in
the capillary-based sub-second mixing device has been previously described.29 (link),30 (link) PerkinElmer Series 200 pumps and autosamplers were used to deliver
solvents. Pepsin (porcine gastric mucosa, Sigma-Aldrich) or Protease
XIII (Aspergillus saitoi, Sigma-Aldrich) was cross-linked in-house
onto NHS-activated agarose (PierceTM, Thermo Fisher Scientific). Digestion
columns were constructed in-house using PEEK tubing with an ID of
0.040” and a 2 μm pore-size frit upstream of the ESI
emitter. All data were acquired on a Waters Synapt G2-S, and IMS was
employed in the TriWave cell to improve the spatial resolution of
peptides in the digested samples. Peptide identification was performed
using Proteome Discoverer (Thermo Fisher Scientific) after LC-MS/MS
analysis with an Orbitrap Elite Hybrid Ion Trap-Orbitrap Mass Spectrometer.
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