Unless noted otherwise, all starting materials and solvents were used as obtained from commercial suppliers (Aldrich, Yongin, Korea) without further purification. Organic solvents used in this study were dried over appropriate drying agents and distilled prior to use. Thin layer chromatography was carried out using Merck silica gel 60 F254 plates, and flash chromatography was performed automatically with Biotage Isolera or manually using Merck silica gel 60 (0.040–0.063 mm, 230–400 mesh, Seoul, Korea). 1H and 13C NMR spectra were recorded using JEOL-500 and BRUKER (Seongnam, Korea) AVANCE-500. 1H and 13C NMR chemical shifts are reported in parts per million (ppm) relative to TMS, with the residual solvent peak used as an internal reference. Low and high resolution mass spectra were obtained with JEOL JMS-700 instrument (Seoul, Korea) and Agilent Q TOF 6530 (Seoul, Korea). 1H NMR data were reported in the order of chemical shift, multiplicity (br, broad signal; s, singlet; d, doublet; t, triplet; q, quartet; quint, quintet; m, multiplet and/or multiple resonances), number of protons, and coupling constant in hertz (Hz). 1H and 13C NMR spectra of compounds 1f-n and 3f-n are available in Supplementary Materials .
Qtof 6530
Manufactured by Agilent Technologies
Sourced in United States, Germany, Japan
The QTOF 6530 is a high-resolution quadrupole time-of-flight (QTOF) mass spectrometer. It is designed to provide accurate and precise mass measurements for a wide range of analytical applications.
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Lab products found in correlation
Merck silica gel 60 f254 plates, by Merck Group (2 mentions)
Merck silica gel 60, by Merck Group (2 mentions)
Cytation 5 plate reader, by Agilent Technologies (2 mentions)
Nadp nadph quantification kit, by Abcam (2 mentions)
1260 hplc system, by Agilent Technologies (2 mentions)
Avance 500, by Bruker (1 mentions)
1260 hplc, by Agilent Technologies (1 mentions)
Masshunter software, by Agilent Technologies (1 mentions)
Eclipse plus c18 rrhd column, by Agilent Technologies (1 mentions)
Standard calibration mix, by Agilent Technologies (1 mentions)
Masshunter qualitative analysis software, by Agilent Technologies (1 mentions)
500 mhz nmr spectrometer, by JEOL (1 mentions)
400 mhz nmr spectrometer, by Bruker (1 mentions)
Voyager de pro maldi tof spectrometer, by Thermo Fisher Scientific (1 mentions)
Vortex genie 2, by Thermo Fisher Scientific (1 mentions)
1290 infinity 2 uplc, by Agilent Technologies (1 mentions)
Acquity uplc protein beh c4 column, by Waters Corporation (1 mentions)
Bioconfirm software, by Agilent Technologies (1 mentions)
Isolera, by Biotage (1 mentions)
Cary 630 ftir spectrometer, by Agilent Technologies (1 mentions)
13 protocols using qtof 6530
1
NMR Spectroscopy Protocol for Organic Compounds
2
Metabolic Profiling of Cell Cultures
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Glucose uptake and lactate secretion rates were determined by using an Agilent Q-TOF (6530) connected to an Agilent 1260 HPLC system (Santa Clara, CA) in negative ionization mode, as described above. 2-HG production flux was measured by quantitating media 2-HG levels over 24 hr. NADPH levels were measured with a NADP+/NADPH Quantification kit (BioVision) and a BioTek Cytation 5 plate reader (Winooski, VT).
3
High-Resolution MS Analysis of Biomolecules
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HPLC-MS experiments were carried out on an Agilent (Santa Clara, CA, USA) Q-TOF 6530 coupled with an Agilent 1260 HPLC in positive ion mode. The mass range was set to 300–1700 amu with auto MS2 acquisition (MS scan rate 2/s, MSMS scan rate 3/s) and active exclusion after three spectra and static exclusion from 300 to 400 m/z. Gas temperature was 300 °C with a gas flow of 11 l min–1 and nebulizer at 35 psig. Collision energies were set to 20 keV. Data were analyzed with MassHunter software (Agilent).
For fourier transform mass spectrometry measurements, samples were injected by a nanomate-electrospray ionization robot (Advion, Ithaca, New York, USA) for consecutive electrospray into the MS inlet of a LTQ 6.4 T Fourier transform ion cyclon resonance (FT-ICR) mass spectrometer (Thermo Finnigan, Thermo Fisher Scientific). MS and MS2 data were acquired in the positive ion mode. Fourier transform mass spectrometry data were acquired in 400–2000 m/z scans. Selected peptide mass signals were manually isolated and fragmented by CID. MSn data were collected either in ion trap or FT detection mode. All data were analyzed using QualBrowser, which is part of the Xcalibur LTQ-FT software package (Thermo Fisher, Waltham, MA, USA).
For fourier transform mass spectrometry measurements, samples were injected by a nanomate-electrospray ionization robot (Advion, Ithaca, New York, USA) for consecutive electrospray into the MS inlet of a LTQ 6.4 T Fourier transform ion cyclon resonance (FT-ICR) mass spectrometer (Thermo Finnigan, Thermo Fisher Scientific). MS and MS2 data were acquired in the positive ion mode. Fourier transform mass spectrometry data were acquired in 400–2000 m/z scans. Selected peptide mass signals were manually isolated and fragmented by CID. MSn data were collected either in ion trap or FT detection mode. All data were analyzed using QualBrowser, which is part of the Xcalibur LTQ-FT software package (Thermo Fisher, Waltham, MA, USA).
4
Analytical Characterization of Natural Compounds
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All substrates were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Samples were routinely analyzed on an Agilent 1260 Infinity II HPLC instrument (Santa Clara, CA, USA). The HPLC was equipped with an Agilent Eclipse Plus C18 RRHD column (1.8 µm, 2.1 mm × 50 mm) and eluted with a linear gradient of 10–95% of acetonitrile-water for 20 min, 95% acetonitrile-water for 10 min and drop down to 10% in 5 min, then 10% acetonitrile-water for 5 min at a flow rate of 0.5 mL/min. 1H-, 13C-, HSQC- and HMBC-NMR spectra were recorded on an Agilent 600 DD2 spectrometer. Chemical shift values (δ) are given in parts per million (ppm) and the coupling constants (J values) are in Hz. Chemical shifts were referenced to the residual solvent peaks of DMSO-d6. HPLC-HRESIMS spectra were acquired on an Agilent 1290 Infinity II HPLC coupled with an Agilent QTOF 6530 instrument operated in negative ion mode using capillary and cone voltages of 3.6 kV and 40–150 V, respectively. For accurate mass measurements the instrument was calibrated each time using a standard calibration mix (Agilent) in the range of m/z 150–1900.
5
Analyzing Intact Proteins by LC-ESI-MS
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Intact proteins were analyzed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) using a QTOF 6530 instrument (Agilent, Waldbronn, Germany). Spectra deconvolution was performed with Agilent MassHunter Qualitative Analysis software employing the maximum entropy deconvolution algorithm.
6
Optimization of NMR and Mass Spectrometry
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Starting materials and solvents were purchased from common commercial sources and used without further purification. Melting points were determined on Fisher melting apparatus and are uncorrected. 1H NMR (500 MHz) and 13 C NMR (125 MHz) spectra were recorded on JEOL a 500 MHz NMR Spectrometer and using DMSO-d6 and CDCl3 as solvents, at Faculty of Science, Mansoura University. Also Bruker 400 MHz NMR Spectrometer at Faculty of Pharmacy, Mansoura University was used. The chemical shift (δ) is reported in ppm, and coupling constants (J) are given in Hz. The HRMS was recorded on Q-TOF, 6530 (Agilent Technologies) at Faculty of pharmacy, Fayoum University. All reactions were monitored by TLC with visualisation by UV irradiation.
7
Mass Spectrometry Analysis of Modified Nisin
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For MALDI–TOF–MS analysis, 1 μl of HPLC-purified sample was applied to the matrix target and treated as described earlier on a Voyager DE Pro MALDI-TOF spectrometer (Applied Biosystems) (van Heel et al., 2013 (link)). Data analysis was carried out with “Data Explorer” software version 4.0.0.0 (Applied Biosystems). Calculation of theoretical masses with ncAA-modified nisin variants was carried out with “massXpert”, version 3.4.0 (Rusconi, 2009 (link)).
For LC–ESI–TOF–MS analysis, IMAC-purified samples were analyzed using a QTOF 6530 instrument (Agilent) as described (Baumann et al., 2017 (link)).
For LC–ESI–TOF–MS analysis, IMAC-purified samples were analyzed using a QTOF 6530 instrument (Agilent) as described (Baumann et al., 2017 (link)).
8
Quantitative Bile Acid Analysis by LC-MS
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Internal standard (13C6-DCF) was added to 25 µl coculture medium to give a final concentration of 20 μM DCF when at 50–100× Cmax (4.4 µM was Cmax for our study), and 2 μM with DCF at 1× Cmax of d5-GCA (0.5–1 μM) was added as an internal standard prior to sample extraction for bile acid measurements. The choice was arbitrary. 4.4 is a low value in the range of the physiologic dose, and higher values were chosen to represent overdosing. MeOH was then added at a 1:4 ratio [(v/v); 25 µl sample/100 µl MeOH). Resulting suspensions were maintained at −20°C for 5 minutes, vortexed for 20 seconds, and subjected to gentle shaking for 5 minutes on a Fisher Vortex Genie 2 with a vortex adapter. The samples were then maintained at −20°C for 5 minutes and centrifuged at 15,000 rpm for 10 minutes. The supernatants were then collected carefully (without disturbing the protein pellet) and dried in a SpeedVac (Savant Instruments, Holbrook, NY). Samples were prepared immediately for liquid chromatography (LC)–mass spectrometry (MS) analysis by resuspension in 2% ACN containing 0.1% FA. Injections of 1–5 µl were analyzed on an Agilent QTOF 6530 using parameters described previously (Sarkar et al., 2015 (link)).
9
Metabolic Profiling of Cell Cultures
10
Methanol Restructuring of Dried Products
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Products were first dried in vacuo and then restructured in methanol. HPLC–HRESI -MS and MS–MS spectra were acquired on an AB SCIEX HPLC coupled with an Agilent QTOF 6530 instrument (capillary: 3.6 kV, cone voltages: 40–150 V). The collision energy was 35 V and was calibrated each time with a standard calibration solution (Agilent) (m/z 150–800).
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