Dried finely pulverized cinnamon specimens (10 mg) were extracted by adding 2 mL 70% MeOH, containing 10 μg mL −1 umbelliferone as an internal standard sonicated for 20 min with frequent shaking, then centrifuged at 12,000× g for 10 min to remove debris. The filtered extract through a 0.22 μm filter was subjected to solid-phase extraction using a C18 cartridge (Sep-Pack, Waters, Milford, MA, USA) as previously described [87 (link)]. Cinnamon bark methanol extracts (2 μL) were injected on an HSS T3 column (100 × 1.0 mm, particle size 1.8 μm; Waters, Milford, MA, USA) installed on an ACQUITY UPLC system (Waters, Milford, MA, USA) equipped with a 6540 Agilent Ultra-High-Definition (UHD) Accurate-Mass Q-TOF-LC-MS (Palo Alto, CA, USA) coupled to an ESI interface, operated in positive or negative ion mode under the exact conditions of our previous work [56 (link)]. Characterization of metabolites was performed using their UV–VIS spectra (220–600 nm), exact masses, in addition to MS2 in both ionization modes, RT data, and reference literature and searching the phytochemical dictionary of natural products [88 ].
C18 cartridge
Manufactured by Waters Corporation
Sourced in United States
The C18 cartridge is a solid-phase extraction (SPE) device used for sample preparation in analytical chemistry. It is designed to selectively retain and separate analytes from complex matrices based on their affinity for the hydrophobic C18 stationary phase.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin, by Promega (7 mentions)
Iodoacetamide, by Merck Group (7 mentions)
Bradford assay, by Bio-Rad (5 mentions)
Mass spectrometry grade trypsin gold, by Promega (3 mentions)
Pngase f, by New England Biolabs (2 mentions)
Acetonitrile, by Merck Group (2 mentions)
Triple quadrupole mass spectrometer, by Waters Corporation (2 mentions)
Formic acid, by Merck Group (2 mentions)
Lys c, by Promega (2 mentions)
Mass spectrometry grade trypsin, by Promega (2 mentions)
Affi prep, by Bio-Rad (2 mentions)
Oasis μelusion plate, by Waters Corporation (2 mentions)
Hss t3 column, by Waters Corporation (1 mentions)
Acquity uplc system, by Waters Corporation (1 mentions)
Quant kit, by GE Healthcare (1 mentions)
125i sodium iodide, by PerkinElmer (1 mentions)
Xbridge c18, by Waters Corporation (1 mentions)
Tracerlab fx mei, by GE Healthcare (1 mentions)
Tripletof 5600 mass spectrometer, by AB Sciex (1 mentions)
Eksigent nanolc 400 system, by AB Sciex (1 mentions)
55 protocols using c18 cartridge
1
Rapid Cinnamon Metabolite Profiling
2
Quantification of Plant Phytohormones and Lipid Peroxidation
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The amount of MDA was measured on the seventh day. IAA, zeatin-type cytokinins, and ABA in plant tissues were determined on the second day after spraying the plants. The youngest leaves were used for chemical tests. To isolate phytohormones, shoots and roots were homogenized and extracted with 80% ethanol. Zeatin and its derivatives (in particular, zeatin riboside) in aqueous residue were concentrated on a C-18 cartridge (Waters Corporation, Milford, MA, USA). After solvent evaporation, the dry residue was dissolved in 0.02 mL of 80% ethanol, and zeatin metabolites were separated using thin-layer chromatography [45 (link)]. Extraction of ABA and IAA from aliquots of aqueous residues was performed with the diethyl ether, according to the modified method, as described by Kudoyarova et al. [46 (link)]. Hormones were immunoassayed using the corresponding specific antibodies.
Membrane lipid peroxidation was assayed as the amount of MDA, a product of lipid peroxidation. Fresh wheat leaves were homogenized in 10% trichloroacetic acid and then centrifuged at 10,000× g rpm. The amount of MDA in the extract was determined by the spectrophotometric method, by reaction with thiobarbituric acid [47 (link)]. Measurements were carried out in three biological and three analytical repetitions.
Membrane lipid peroxidation was assayed as the amount of MDA, a product of lipid peroxidation. Fresh wheat leaves were homogenized in 10% trichloroacetic acid and then centrifuged at 10,000× g rpm. The amount of MDA in the extract was determined by the spectrophotometric method, by reaction with thiobarbituric acid [47 (link)]. Measurements were carried out in three biological and three analytical repetitions.
3
Protein Reduction, Alkylation, and Trypsin Digestion
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Proteins were
reduced (10 mM DTT, 56 °C for 45 min), alkylated (50 mM iodoacetamide,
room temperature in the dark for 1 h), and excess iodoacetamide was
quenched with DTT to a final concentration of 25 mM. Proteins were
subsequently digested with trypsin (sequencing grade, Promega), at
an enzyme/substrate ratio of 1:100, at room temperature overnight
in 100 mM ammonium acetate, pH 8.9. trypsin activity was quenched
by adding formic acid to a final concentration of 5%. Urea was removed
from the samples by reverse phase desalting using a C18 cartridge
(Waters) and peptides were lyophilized and stored at −80 °C.
reduced (10 mM DTT, 56 °C for 45 min), alkylated (50 mM iodoacetamide,
room temperature in the dark for 1 h), and excess iodoacetamide was
quenched with DTT to a final concentration of 25 mM. Proteins were
subsequently digested with trypsin (sequencing grade, Promega), at
an enzyme/substrate ratio of 1:100, at room temperature overnight
in 100 mM ammonium acetate, pH 8.9. trypsin activity was quenched
by adding formic acid to a final concentration of 5%. Urea was removed
from the samples by reverse phase desalting using a C18 cartridge
(Waters) and peptides were lyophilized and stored at −80 °C.
4
Serum Protein Profiling using MARS
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The concentrations of individual serum proteins from the multiple affinity removal system column were measured using a Quant kit (GE Healthcare, Piscataway, NJ, USA). Protein samples (1 mg) were treated with 5-mM Tris (2-carboxyethyl) phosphine (Pierce, Rockford, IL, USA), and then incubated at 37℃ at 75×g for 30 min for reduction. Next, 15-mM iodoacetamide (Sigma-Aldrich, St. Louis, MO, USA) was added to each sample and incubated at room temperature at 300 rpm for 1 h in the dark. Then, samples were incubated overnight at 37℃ with trypsin (Promega, Madison, WI, USA) to digest the proteins into peptides. The C18 cartridge (Waters, Milford, MA, USA) was used to clean up the peptide mixtures. The dried peptides were dissolved in 360 µL of water for OFFGEL electrophoresis.
5
Radiosynthesis of 125I-labeled ITdU
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Chemicals and solvents were purchased
from Sigma-Aldrich (St. Louis, MI, USA) and Merck (Darmstadt, Germany)
or otherwise as indicated. No-carrier-added (n.c.a.) sodium [125I]iodide was obtained from PerkinElmer (Waltham, MA, USA).
No-carrier-added Na125I was used as received. The 5-tributylstannyl
precursor of ITdU was synthesized according to previously reported
methods and provided by PD Dr. rer. nat. Boris Zlatopolskiy.15 (link) For labeling, 3 μL of a ITdU precursor
solution (123 mM in 66% MeOH aq) was added to 17 μL of buffer
(0.2 M phosphate buffer pH 2, 30% MeOH aq). Then, 10 μL of a
Na125I solution in 0.05 M NaOH was added before addition
of 16 μL Chloramine T solution (0.66 mg/mL in 66% methanol).
After 10 min at room temperature, the reaction mixture was purified
via high performance liquid chromatography (HPLC) on an analytical
C4 column (Multochrom-100-5 μ-C4, 250 mm × 4 mm, CS-Chromatographie,
Langerwehe, Germany). Quality control was performed by the same HPLC
method. After purification, the product fraction was concentrated
by dilution and sorption by a C18 cartridge (#WAT023501, Waters, Milford,
MA, USA), followed by rinsing, elution with 1 mL of MeCN, evaporation,
and dissolution in EtOH. Overall radiochemical yield (RCY) was >70%,
and final radiochemical purity was >95%. Mean product molar activities
were Am = 41 GBq/μmol.
from Sigma-Aldrich (St. Louis, MI, USA) and Merck (Darmstadt, Germany)
or otherwise as indicated. No-carrier-added (n.c.a.) sodium [125I]iodide was obtained from PerkinElmer (Waltham, MA, USA).
No-carrier-added Na125I was used as received. The 5-tributylstannyl
precursor of ITdU was synthesized according to previously reported
methods and provided by PD Dr. rer. nat. Boris Zlatopolskiy.15 (link) For labeling, 3 μL of a ITdU precursor
solution (123 mM in 66% MeOH aq) was added to 17 μL of buffer
(0.2 M phosphate buffer pH 2, 30% MeOH aq). Then, 10 μL of a
Na125I solution in 0.05 M NaOH was added before addition
of 16 μL Chloramine T solution (0.66 mg/mL in 66% methanol).
After 10 min at room temperature, the reaction mixture was purified
via high performance liquid chromatography (HPLC) on an analytical
C4 column (Multochrom-100-5 μ-C4, 250 mm × 4 mm, CS-Chromatographie,
Langerwehe, Germany). Quality control was performed by the same HPLC
method. After purification, the product fraction was concentrated
by dilution and sorption by a C18 cartridge (#WAT023501, Waters, Milford,
MA, USA), followed by rinsing, elution with 1 mL of MeCN, evaporation,
and dissolution in EtOH. Overall radiochemical yield (RCY) was >70%,
and final radiochemical purity was >95%. Mean product molar activities
were Am = 41 GBq/μmol.
6
Radiolabeling of 4-amino-3-hydroxypyridine
7
2-ClHDyA Proteome Enrichment
8
Quantification of Plant Hormones
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To determine the content of hormones, a sample of 5 plants was taken for each biological replicate (roots or shoots) on the sixth and eleventh days after the start of experiments. Hormones were extracted with 80% ethanol (1:10) overnight. Extract separated by filtration was evaporated to an aqueous residue and divided into two parts for determination of cytokinins and indoleacetic acid (IAA, a hormone from the class of auxins). Cytokinins from the aqueous residue after their concentration on a C-18 cartridge (Waters Corporation, Milford, MA, USA) were separated by thin layer chromatography as previously described [31 (link)]. Contents of zeatin, its riboside and nucleotide in the corresponding chromatographic zones were determined by enzyme-linked immunosorbent assay (ELISA) using antibodies against zeatin riboside [32 (link)]. IAA was partitioned from the acidified aqueous residue with diethyl ether as previously described [28 (link)]. After methylation of samples, the IAA concentration in the extract was determined by ELISA using the appropriate specific antibodies [33 (link)].
9
Serum Protein Characterization by Mass Spectrometry
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Concentration of serum proteins was measured and 100 μg protein samples were prepared for mass analysis. Proteins were reduced by treatment with 5 mM Tris (2-carboxyethyl) phosphine (Pierce, Rockford, IL, USA) at 37 °C, 300 rpm, for 30 min. For alkylation, 15 mM Iodoacetamide (Sigma-Aldrich, St. Louis, MO, USA) was added to the samples at 25 °C, with agitation at 300 rpm for 1 h in the dark. Proteins were cleaved into peptides, using trypsin, overnight at 37 °C. Mass spectrometry-grade trypsin gold (Promega, Madison, WI, USA) was used for the purpose. After a sample containing chemical reagents was cleaned by a C18 cartridge (Waters, Milford, MA, USA), it was separated based on isoelectric point using the OFFGEL Fractionator with a 12-well setup (3100 OFFGEL Low Res Kit, pH 3–10; Agilent Technologies, Santa Clara, CA, USA). To identify the proteins, the peptide fraction that was separated into 12 samples was analyzed using a TripleTOF 5600 mass spectrometer (AB SCIEX, Concord, ON, Canada) combined with an Eksigent nanoLC 400 system and cHiPLC® (AB SCIEX, Concord, ON, Canada).
10
Recombinant SARS-CoV-2 S1 Protein Characterization
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All recombinant SARS-CoV-2 S1 proteins were His tagged. S1-CHO and S1-HEK-F were provided, as gifts, by our collaborator AnyGo Technology (Beijing, China), and produced in CHO and human embryonic kidney (HEK) 293F cells, using the Gibco method and the cell-specific expression kits, ExpiCHO and Expi293F (ThermoFisher, Waltham, MA, USA), respectively. S1-BII and S1-HEK-E were produced in baculovirus-infected insect (BII) and HEK 293-EBNA1 cells, respectively, and were purchased from Sino Biological (Beijing, China). Recombinant Fc-tagged human ACE2 and mouse anti-human IgG1-Fc were from Sino Biological. DL-dithiothreitol (DTT), iodoacetamide (IAA), 2,5-dihydroxybenzoic acid (DHB), ammonium bicarbonate and sodium hydroxide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tween-20 and Dulbecco’s phosphate buffered saline (DPBS) were purchased from Sinopharm. (Shanghai, China). Trypsin was from Promega (Madison, WI, USA). PNGase F was obtained from New England Biolabs (Ipswich, MA, USA). C18 cartridge and 96-well plate were purchased from Waters (Milford, MA, USA). All the chemicals were of analytical grade or better, and used as received without further purification.
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