Optical rotations were measured on Rudolph research analytical AUTOPOL I. The ultraviolet spectra were obtained from a Shimadzu UV-1800 spectrophotometer using MeOH as the solvent. Electronic circular dichroism (ECD) spectra were obtained on a JASCO J-1500 circular dichroism. The infrared (IR) spectra were acquired from a Bruker Vector 22. NMR spectra were recorded on a Bruker AVIII 500 MHz and JEOL 600 Hz, using TMS as the internal standard. HRESIMS spectra were obtained from an Agilent 6224 TOF LC/MS. Analytical HPLC was performed on an Agilent 1260 system using a C18 (Cosmosil, 5 μm, 4.6 × 250 mm) column. The column chromatography was carried out using Silica gel (200–300 mesh, Qing Dao Hai Yang Chemical Group Co., Qing Dao, China).
6224 tof lc ms
Manufactured by Agilent Technologies
Sourced in United States
The 6224 TOF LC/MS is a high-performance liquid chromatography-time of flight mass spectrometry (LC/TOF-MS) system. It provides accurate mass measurement and high-resolution analysis of a wide range of compounds. The system integrates a liquid chromatography unit and a time-of-flight mass spectrometer to enable efficient separation, detection, and identification of analytes.
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Lab products found in correlation
8 protocols using 6224 tof lc ms
1
Spectroscopic Characterization of Organic Compounds
2
HRMS Analysis of Jeffamine Standard
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HRMS experiments
were conducted either on an Agilent 6224 TOF LC/MS [O-time-of-flight
(TOF)] interfaced with the Direct Analysis in Real Time (DART) source
(IonSense DART-100) or on a Bruker MaXis TOF MS (Q-TOF) interfaced
with the DART source (IonSense DART-SVP). A standard of Jeffamine
was used as an internal standard calibration for HRMS DART experiments
carried out in the positive mode. Combustion elemental analysis was
carried out by ALS Environmental located in Tucson, AZ.
were conducted either on an Agilent 6224 TOF LC/MS [O-time-of-flight
(TOF)] interfaced with the Direct Analysis in Real Time (DART) source
(IonSense DART-100) or on a Bruker MaXis TOF MS (Q-TOF) interfaced
with the DART source (IonSense DART-SVP). A standard of Jeffamine
was used as an internal standard calibration for HRMS DART experiments
carried out in the positive mode. Combustion elemental analysis was
carried out by ALS Environmental located in Tucson, AZ.
3
Metabolite Extraction and Quantification
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For metabolite extraction, 450 μl MilliQ water containing internal standards (methionine sulfone and camphor-10-sulfonic acid), 500 l CHCl3, and 250 μl methanol were added to 30 mg tissue or 300 pancreatic islets, and the mixture was homogenized by a bead mill homogenizer. After centrifugation, the supernatant was collected, filtered using a 5-kDa cutoff filter, and freeze-dried. The hydrophilic metabolites were subjected to capillary electrophoresis-mass spectrometry on an Agilent capillary electrophoresis coupled to time-of-flight mass spectrometry (7100 CE and 6224 TOF LC/MS). Metabolite concentrations were calculated as the ratio between each metabolite in the sample and the standard compounds.
4
Quantification of Iron and Bacterial Biomass
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Fe2+ was determined colorimetrically with o-phenanthroline (Tamura et al., 1974 (link)), Fe3+ spectrophotometrically at 300 nm (Mandl and Novakova, 1993 (link)), and total iron by ICP spectrometry (iCAP 6500, Thermo Fisher Scientific, Waltham, MA, United States). The number of bacteria was determined using a Cyrus chamber and an optical microscope BX50 (Olympus, Tokyo). Tetrathionate was determined by mass spectrometry (6224 TOF LC/MS, Agilent Technologies, Santa Clara, CA, United States). Cellular ATP content of planktonic and attached cells was determined as previously described using a bioluminescence assay (Pakostova et al., 2013a (link)). Sufficient amount of attached cells for ATP determination were obtained using the detachment protocol developed previously for cells growing with elemental sulfur (Pakostova et al., 2013b (link)). Differences in the ATP contents were tested by the t-test.
5
Synthesis of Substituted Dipyrromethanes
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All reactions were performed under N2 atmosphere. All reagents and solvents were of reagent grade. The NMR spectra were recorded in CDCl3 and THF-d8 on a Bruker AV Ultra Shield 400 MHz instrument. Absorption spectra were obtained with PG T80. NMR data are represented as follows: chemical shift (ppm), multiplicity (s = singlet, brs = broad singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constants in hertz (Hz). IR spectra were recorded on FTIR spectrometer (Thermo Scientific, Nicolet IS10). HRMS were measured in ESI mode and the mass analyzer of the HRMS was TOF (Agilent 6224 TOF LC–MS). Flash column chromatography was performed on silica gel (230–400 mesh). 5-Substituted dipyrromethanes [35 (link)] and 1,9-diformyldipyrromethanes [36 (link)] were prepared according to literature methods and their spectral data matched literature values.
6
Quantitative Metabolome Analysis by CE-MS
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Quantitative metabolome analysis was carried out by CE-MS. In brief, after the removal of feeder cells, 4 9 10 6 ESCs or iPSCs were cultured for an additional 40 min. Subsequently, these cells were washed three times with ice-chilled KRB and immediately quenched with liquid nitrogen. For metabolite extraction, 450 lL of MilliQ water containing internal standards (5 lM of methionine sulfone and camphor-10-sulfonic acid) was added to the cells, and the mixture was homogenized by a bead mill homogenizer. Then, 500 lL of CHCl 3 and 250 lL of methanol were added to the mixture, and the mixture was homogenized again. After centrifugation, the supernatant containing hydrophilic primary metabolites was collected, filtered by a 5-kDa cutoff filter and dried by freeze drying. The dried hydrophilic metabolites were reconstituted with 25 lL of MilliQ water and then subjected to metabolome analysis using CE-MS (Agilent 7100 CE and 6224 TOF LC/MS). After calculation by using the ratio between each metabolite in the sample and standard compounds, metabolite concentrations were adjusted to the total amount of DNA.
7
Synthesis and Characterization of Novel Compounds
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The chemicals and all solvents used in this work were purchased from the supplier and were used without purification. All reactions were monitored by TLC (Silica gel, aluminum sheets 60 F254, Merck), which were visualized under UV light. and column chromatography was carried out on silica gel 60N (spherical, neutral). Melting points (not corrected) were recorded in open capillary tubes on Electrothermal 9200 digital melting point apparatus (Yozgat Bozok Unıversity). Fourier transform infrared spectra were recorded in the spectral range of 4000-400 cm -1 on a Perkin Elmer Spectrum Two Model FT-IR spectrophotometer using ATR method with a resolution of 4 cm -1 at room temperature (Yozgat Bozok University). HRMS analyses spectra were obtained using a Agilent Technologies 6224 TOF LC/MS (Bilkent University-UNAM). 1 H NMR and 13 C NMR spectra were recorded on a Bruker Ultrashield NMR spectrometer using DMSO-d6 as solvent and TMS as an internal reference (Erciyes University, TAUM Laboratory. Chemical shifts are expressed in δ ppm and coupling constants (J values) are reported in hertz. Multiplicities are expressed as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and br (broad).
8
Usp7 Inhibitor Binding Kinetics
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To initiate reaction, a two-fold excess of compound was incubated with Usp7 mutants in PBS buffer with 0.5mM TCEP. At 0,10,20,30, and 40 minutes, 1.0mg protein was injected over a 6224 TOF LC/MS (Agilent). Mass spectrometry data were analyzed using MassHunger Qualitative Analysis B.06.00 (Agilent). Peaks corresponding to the correct mass for Usp7 and Usp7 + inhibitor were integrated and plotted in GraphPad Prism as a function of time.
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