Target compounds (1 –5 ) were synthesized in our laboratory. The purity of target compounds (1 –5 ) was ≥ 95% using HPLC analysis (Agilent Technologies 1260, Pittsburgh, PA, USA). HPLC-grade methanol and acetonitrile were purchased from Xinkeao Scientific and Technology Co., Ltd. (Beijing, China). CCK-8 and Griess kits were purchased from Promega Corporation (Madison, WI, USA). TNF-α and IL-1β ELISA kits were purchased from Proteintech (Rosemont, IL, USA). The COX enzyme immunoassay kit was purchased from Cayman Chemical (Ann Arbor, MI, USA). Other chemicals and reagents were of analytical grade and commercially available. All chemicals and reagents were used without further purification unless otherwise stated. The reaction was monitored using thin layer chromatography (TLC) and a silica-gel-coated glass plate (Qingdao Ocean Chemical Co., Qingdao, China), and visualized under UV light (254 nm). An FTIR spectrum was carried out on IRPrestige-21, SHIMADZU. MS was recorded using Q-TOF SYNAPT G2 HDMS, Waters. 1H-NMR and 13C-NMR were recorded on a Bruker AVANCE 500 NMR spectrometer (Fällanden, Switzerland) and chemical shifts were reported in ppm. Melting point was recorded on an SGW®X-4 microscope melting apparatus (Shanghai Scientific and Technology Co., Ltd., Shanghai, China). NMR, FTIR, UV/Vis and MS spectra of target compounds are given in Supplementary Materials .
Synapt g2 hdms q tof
Manufactured by Waters Corporation
Sourced in United States
The SYNAPT G2 HDMS Q-TOF is a high-resolution quadrupole time-of-flight mass spectrometer designed for advanced analytical applications. It combines a quadrupole mass filter, a high-definition ion mobility separator, and a time-of-flight mass analyzer to provide high-resolution mass analysis and ion mobility separation capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Masslynx software, by Waters Corporation (2 mentions)
Avance 500 nmr spectrometer, by Bruker (1 mentions)
Milli q advantage a10 ultra pure water purifier, by Merck Group (1 mentions)
Acquity uplc liquid chromatography system, by Waters Corporation (1 mentions)
Acquity ultra performance liquid chromatography system, by Waters Corporation (1 mentions)
Gemini nx rp 18 column, by Phenomenex (1 mentions)
Uplc beh c18 column, by Waters Corporation (1 mentions)
300 mhz spectrometer, by Bruker (1 mentions)
Minidawn treos light scattering detector, by Wyatt Technology (1 mentions)
5 μm mixed c gel permeation columns, by Wyatt Technology (1 mentions)
High performance liquid chromatography (hplc), by Agilent Technologies (1 mentions)
Masslynx software version 4, by Waters Corporation (1 mentions)
Acquity uplc beh 300, by Waters Corporation (1 mentions)
8 protocols using synapt g2 hdms q tof
1
Synthesis and Characterization of Target Compounds
2
Daosheng Four Diagnostic Instrument Protocol
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DS01-A Daosheng Four Diagnostic instrument (Daosheng, China), Waters Acquity UPLC Liquid Chromatography System, Q-TOF SYNAPT G2 HDMS (Waters Corporation, USA), high-speed refrigeration centrifuge (Termo Scientific, Germany), SpeedVac®SPD131Centrifuge enrichment system (Termo Scientific, Germany), VORTEX GENIUS 3 VORTEX oscillator (IKA, Germany), and Milli-Q Advantage A10 Ultra-pure Water Purifier (Milli-pore, USA).
3
Quantitative EPO Glycopeptide Analysis
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First, 250 μg
of each EPO sample was denatured with 0.4 M Tris and 8 M Gua-HCl,
pH 8.5. Next, samples were reduced with 21 mM DTT at 50 °C for
60 min and then alkylated with 50 mM IAA at room temperature (RT)
for 30 min in the dark. Prior to Glu-C digestion, samples were buffer-exchanged
to 50 mM aqueous ammonium hydrogen carbonate, pH 7.8, with NAP-5 columns.
Samples were then digested with GluC at 25 °C for 16–18
h at a 1:20 enzyme/protein ratio.
Five micrograms of digested
EPO was loaded on a Waters Corp. (Milford, MA, USA) ACQUITY ultraperformance
liquid chromatography system and C8 ethylene bridged hybrid columns
(2.1 mm × 150 mm, 1.7 μm, 130 Å) coupled to Q-TOF
Synapt G2 HDMS by Waters, Q-TOF by Bruker Corporation (Billerica,
MA, USA), or Orbitrap Velos or Fusion from Thermo Fisher Scientific.
The column was kept at 65 °C and ran as a 65 min gradient method
with 0.1% FA in H2O (solvent A) and 0.1% FA in ACN (solvent
B) applied at a flow rate of 300 μL/min (0–30 min: 1–20%
B, 30–60 min: 20–5% B, 60–65 min: 35–60%
B, 65–75 min: 80–99% B, 75–80 min: 1% B). MS
measurements were performed in positive ion mode, and data were acquired
in the 500–2000 m/z range.
Extracted ion chromatograms were generated for m/z values of expected glycopeptides; peaks were integrated,
and the resulting areas were used to obtain relative glycan abundance
at each site.
of each EPO sample was denatured with 0.4 M Tris and 8 M Gua-HCl,
pH 8.5. Next, samples were reduced with 21 mM DTT at 50 °C for
60 min and then alkylated with 50 mM IAA at room temperature (RT)
for 30 min in the dark. Prior to Glu-C digestion, samples were buffer-exchanged
to 50 mM aqueous ammonium hydrogen carbonate, pH 7.8, with NAP-5 columns.
Samples were then digested with GluC at 25 °C for 16–18
h at a 1:20 enzyme/protein ratio.
Five micrograms of digested
EPO was loaded on a Waters Corp. (Milford, MA, USA) ACQUITY ultraperformance
liquid chromatography system and C8 ethylene bridged hybrid columns
(2.1 mm × 150 mm, 1.7 μm, 130 Å) coupled to Q-TOF
Synapt G2 HDMS by Waters, Q-TOF by Bruker Corporation (Billerica,
MA, USA), or Orbitrap Velos or Fusion from Thermo Fisher Scientific.
The column was kept at 65 °C and ran as a 65 min gradient method
with 0.1% FA in H2O (solvent A) and 0.1% FA in ACN (solvent
B) applied at a flow rate of 300 μL/min (0–30 min: 1–20%
B, 30–60 min: 20–5% B, 60–65 min: 35–60%
B, 65–75 min: 80–99% B, 75–80 min: 1% B). MS
measurements were performed in positive ion mode, and data were acquired
in the 500–2000 m/z range.
Extracted ion chromatograms were generated for m/z values of expected glycopeptides; peaks were integrated,
and the resulting areas were used to obtain relative glycan abundance
at each site.
4
Spectroscopic Analysis of Natural Compounds
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Ultraviolet lamp (UVP, LLC, USA) was used for visualization of spots on thin layer chromatograms at 254 and/or 365 nm. UV spectra of different samples were acquired using a Spectronic® GenesysTM 2PC UV spectrophotometer (Shimazdu, Japan) as solutions in methanol as well as with different diagnostic UV shift and complexing reagents for flavonoids [20 ]. 1H (400 MHz) as well as 13C NMR (100 MHz) and distortionless enhancement by polarization transfer (DEPT-Q; 100 MHz) spectra were recorded on Bruker Avance 400 MHz instruments in DMSO-d6 and CD3OD. Chemical shift values (δ) were recorded in ppm units and coupling constants (J) in Hz. Solvent signals of DMSO-d6 (δH 2.5 ppm and δC 39.5 ppm) and CD3OD (δH 3.3 ppm and δC 49.0 ppm) were considered as the internal reference signals for calibration. Electrospray ionization mass spectrometry (ESI‒MS) spectra were obtained using a Synapt G2 HDMS QTOF (quadrupole time-of-flight)-mass spectrometer (Waters, Germany). HPLC separations and purifications were performed on KNAUER HPLC (smart line pump 1000, degasser, diode array detector) with UV Detector, using semi-prep RP-18 column (5 μm, 10 × 250 mm; Waters XBridge, Germany), while an analytical Gemini-NX RP-18 column (5 μm, 4.60 × 100 mm; Phenomenex, Germany) was used for analytical purposes.
5
LC-ESI-MS/MS Analysis of Peptides
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LC-ESI-MS/MS was carried out
on a Waters iClass HPLC system using a Waters UPLC BEH C18 column
(1.0 mm × 150 mm, 1.7 μm), and the column temperature was
set to 40 °C. Mobile phase [A]: H2O + 0.1% formic
acid (v/v); [B]: acetonitrile + 0.1% formic acid (v/v). Starting with
isocratic conditions of 5% [B] for 1 min followed by the gradient
5% [B] to 40% [B] over 10 min and 40% [B] to 95% [B] over 6 min. The
flow rate was set to 80 μL/min. Detection was performed first
on a UV detector at 214 and 280 nm (TUV, Waters iClass) followed by
a Synapt G2 HDMS QTof (Waters, Manchester, UK) equipped with an ESI
source and adjusted with common voltages for MS peptide analysis.
The collision energy for low-energy trace was switched off and ramped
from 16 V up to 42 V for the high-energy trace. The data were acquired,
processed, and analyzed in Waters MassLynx software.
on a Waters iClass HPLC system using a Waters UPLC BEH C18 column
(1.0 mm × 150 mm, 1.7 μm), and the column temperature was
set to 40 °C. Mobile phase [A]: H2O + 0.1% formic
acid (v/v); [B]: acetonitrile + 0.1% formic acid (v/v). Starting with
isocratic conditions of 5% [B] for 1 min followed by the gradient
5% [B] to 40% [B] over 10 min and 40% [B] to 95% [B] over 6 min. The
flow rate was set to 80 μL/min. Detection was performed first
on a UV detector at 214 and 280 nm (TUV, Waters iClass) followed by
a Synapt G2 HDMS QTof (Waters, Manchester, UK) equipped with an ESI
source and adjusted with common voltages for MS peptide analysis.
The collision energy for low-energy trace was switched off and ramped
from 16 V up to 42 V for the high-energy trace. The data were acquired,
processed, and analyzed in Waters MassLynx software.
6
LED-Powered Photochemical Reactor Setup
7
Mass Spectrometry Analysis of EF-Tu and pEF-Tu
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EF-Tu and pEF-TuT382 samples were prepared at 10 μM protein concentration in 100 mM ammonium acetate buffer (pH 6.9). Both samples were introduced into the vacuum of the mass spectrometer using nanoelectrospray ionization with in-house–prepared gold-coated borosilicate glass capillaries with a voltage of approximately +1.4 kV. Spectra were recorded on a SYNAPT G2 HDMS Q-TOF (Waters) instrument. Critical voltages throughout the instrument were 50, 1, 25, 45, and 0.5 V for the sampling cone, extraction cone, trap collision voltage, trap dc bias, and transfer collision energy, respectively. Pressure throughout the instrument was 2.87 mbar for the source, 3.22 × 10−2 mbar for the trap collision cell, and 3.23 × 10−2 for the transfer collision cell. Mass spectra were externally calibrated using CsI, and the mass spectrometry data were analyzed using the MassLynx software version 4.1 (Waters).
8
Lipopeptide Characterization by UPLC-MS
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The residue obtained from lipopeptide extraccion was dissolved in 10% methanol and analysed by high-pressure liquid chromatography (UPLC) (Acquity UPLC® BEH300, Waters) coupled to a high definition mass spectrometry (SYNAPT G2 HDMS Q-TOF. Waters). Mass spectrometry was carried out by positive ionisation electrospray (ESI+). The obtained data were processed by the MassLynxTM software (Waters).
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