agilent 6224 tof lc ms system, by Agilent Technologies - Product details

1

Enzymatic Characterization of GlcNAc Oligosaccharide

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For this experiment, 10 mm (GlcNAc)₃ was treated with 10 μmBmCDA8 in 20 mm Tris (pH 7.4) and 20 mm NaCl at 30 °C for 48 h. The samples were boiled at 100 °C for 2 min and then centrifuged at 17,000 × g for 10 min. Purified OfHex1 (51 (link)) was added to the samples to a final concentration of 10 μm. The resulting solution was incubated at 30 °C for 48 h. NaN₃ was added to all the samples at a final concentration of 0.03% to prevent bacteria growth. The reactions were terminated by boiling for 2 min. The samples treated with and without OfHex1 were subjected to ESI-MS and recorded in both positive and negative mode using an Agilent 6224 TOF LC/MS system with a dual-nebulizer ESI source (Agilent Technology).

Liu L., Zhou Y., Qu M., Qiu Y., Guo X., Zhang Y., Liu T., Yang J, & Yang Q. (2019). Structural and biochemical insights into the catalytic mechanisms of two insect chitin deacetylases of the carbohydrate esterase 4 family. The Journal of Biological Chemistry, 294(15), 5774-5783.

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2

CE-TOFMS-based Metabolome Profiling

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Metabolome analysis was performed as previously described^{25 (link)}. Frozen collected samples were thawed and centrifuged at 13,000×g for 5 min at 4 °C. The supernatant was transferred to a 5-kDa-cutoff filter (Human Metabolome Technologies, Tsuruoka, Japan) to remove proteins of sizes greater than 5 kDa. Prior to CE-TOFMS analysis, a 45 µL aliquot of the filtrate was added to 5 µL of Milli-Q water containing reference compounds (200 mmol/L each of methionine sulfone, D-camphor-10-sulfonic acid, 3-aminopyrrolidine, and trimesic acid). CE-TOFMS-based metabolome profiling was performed using an Agilent 7100 Capillary Electrophoresis system (Agilent technologies, Waldbronn, Germany), an Agilent 6224 TOF LC/MS system (Agilent technologies, Santa Clara, CA), an Agilent 1200 series isocratic HPLC pump, a G1603A Agilent CE-MS adapter kit, and a G1607A Agilent CE-electrospray ionization (ESI)-MS sprayer kit. In anionic metabolites analysis, ESI sprayer was replaced with a platinum needle instead of an initial stainless-steel needle^{25 (link)}. Other conditions of the CE–ESI–MS sprayer were the same as received. The metabolome analysis conditions were the same as those described elsewhere^{9 (link),26 (link)–28 (link)}. Data analysis were performed using the metabolome analysis software MasterHands as previously described^{29 (link)}.

Maruyama Y., Nishimoto Y., Umezawa K., Kawamata R., Ichiba Y., Tsutsumi K., Kimura M., Murakami S., Kakizawa Y., Kumagai T., Yamada T, & Fukuda S. (2022). Comparison of oral metabolome profiles of stimulated saliva, unstimulated saliva, and mouth-rinsed water. Scientific Reports, 12, 689.

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3

Metabolomic Analysis of Stable Isotope Labeling in Cell Lines

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All stable isotopes were purchased from Cambridge Isotope Laboratories, Inc. Cells were seeded on a 100 mm dish and cultured overnight. On the next day, culture media were replaced to [U-¹³C] glucose containing culture media (DMEM/10% FBS/4.5 g/L [U-¹³C] glucose for U87MG, MEM/10% HS/1 g/L [U-¹³C] glucose for primary glia) or ¹⁵N₅-guanosine-containing culture media (DMEM/10% dialyzed FBS or MEM/10% HS/1 μM guanosine). For neurospheres, neural stem cell-supporting culture medium (NSM) containing 17.5 mM [U-¹³C] glucose, 2.5 mM ¹³C₅, ¹⁵N₂-glutamine was used. Cells were harvested at indicated times. Extraction of metabolites was performed as previously described⁶². Capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) experiments were performed using an Agilent G7100A Capillary Electrophoresis instrument (Agilent technologies), an Agilent 6224 TOF LC/MS system (Agilent technologies), an Agilent 1200 series isocratic HPLC pump, a G1603A Agilent CE-MS adapter kit and a G1607A Agilent CE-electrospray ionization (ESI)-MS sprayer kit. For CE-TOFMS system control and data acquisition, we used an Agilent MassHunter software. Detailed conditions of CE-TOFMS-based metabolome analysis were described^{63 ,64}. Capillary ion chromatography (IC)-MS analysis was performed using a Dionex ICS-5000⁺ system equipped with a Q Exactive Orbitrap MS system (Thermo Fisher Scientific).

Kofuji S., Hirayama A., Eberhardt A.O., Kawaguchi R., Sugiura Y., Sampetrean O., Ikeda Y., Warren M., Sakamoto N., Kitahara S., Yoshino H., Yamashita D., Sumita K., Wolfe K., Lange L., Ikeda S., Shimada H., Minami N., Malhotra A., Morioka S., Ban Y., Asano M., Flanary V.L., Ramkissoon A., Chow L.M., Kiyokawa J., Mashimo T., Lucey G., Mareninov S., Ozawa T., Onishi N., Okumura K., Terakawa J., Daikoku T., Wise-Draper T., Majd N., Kofuji K., Sasaki M., Mori M., Kanemura Y., Smith E.P., Anastasiou D., Wakimoto H., Holland E.C., Yong W.H., Horbinski C., Nakano I., DeBerardinis R.J., Bachoo R.M., Mischel P.S., Yasui W., Suematsu M., Saya H., Soga T., Grummt I., Bierhoff H, & Sasaki A.T. (2019). IMP dehydrogenase-2 drives aberrant nucleolar activity and promotes tumorigenesis in glioblastoma. Nature cell biology, 21(8), 1003-1014.

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4

Metabolomic Analysis of Stable Isotope Labeling in Cell Lines

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All stable isotopes were purchased from Cambridge Isotope Laboratories, Inc. Cells were seeded on a 100 mm dish and cultured overnight. On the next day, culture media were replaced to [U-¹³C] glucose containing culture media (DMEM/10% FBS/4.5 g/L [U-¹³C] glucose for U87MG, MEM/10% HS/1 g/L [U-¹³C] glucose for primary glia) or ¹⁵N₅-guanosine-containing culture media (DMEM/10% dialyzed FBS or MEM/10% HS/1 μM guanosine). For neurospheres, neural stem cell-supporting culture medium (NSM) containing 17.5 mM [U-¹³C] glucose, 2.5 mM ¹³C₅, ¹⁵N₂-glutamine was used. Cells were harvested at indicated times. Extraction of metabolites was performed as previously described⁶². Capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) experiments were performed using an Agilent G7100A Capillary Electrophoresis instrument (Agilent technologies), an Agilent 6224 TOF LC/MS system (Agilent technologies), an Agilent 1200 series isocratic HPLC pump, a G1603A Agilent CE-MS adapter kit and a G1607A Agilent CE-electrospray ionization (ESI)-MS sprayer kit. For CE-TOFMS system control and data acquisition, we used an Agilent MassHunter software. Detailed conditions of CE-TOFMS-based metabolome analysis were described^{63 ,64}. Capillary ion chromatography (IC)-MS analysis was performed using a Dionex ICS-5000⁺ system equipped with a Q Exactive Orbitrap MS system (Thermo Fisher Scientific).

Kofuji S., Hirayama A., Eberhardt A.O., Kawaguchi R., Sugiura Y., Sampetrean O., Ikeda Y., Warren M., Sakamoto N., Kitahara S., Yoshino H., Yamashita D., Sumita K., Wolfe K., Lange L., Ikeda S., Shimada H., Minami N., Malhotra A., Morioka S., Ban Y., Asano M., Flanary V.L., Ramkissoon A., Chow L.M., Kiyokawa J., Mashimo T., Lucey G., Mareninov S., Ozawa T., Onishi N., Okumura K., Terakawa J., Daikoku T., Wise-Draper T., Majd N., Kofuji K., Sasaki M., Mori M., Kanemura Y., Smith E.P., Anastasiou D., Wakimoto H., Holland E.C., Yong W.H., Horbinski C., Nakano I., DeBerardinis R.J., Bachoo R.M., Mischel P.S., Yasui W., Suematsu M., Saya H., Soga T., Grummt I., Bierhoff H, & Sasaki A.T. (2019). IMP dehydrogenase-2 drives aberrant nucleolar activity and promotes tumorigenesis in glioblastoma. Nature cell biology, 21(8), 1003-1014.

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Analytical Techniques for Reaction Mixture

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Quantitative analyses of reaction mixtures were preformed on HPLC-DAD instrument (Agilent, Santa Clara, CA, USA) with an external standard of IPTC or 1.
For qualitative analyses of the reaction products, several instruments were used. GC and GC/MS analyses were carried out on HP 6890 instrument (Hewlett Packard, Palo Alto, CA, USA), equipped with HP5 (30 m × 0.32 mm i.d.) column and FID or quadrupole MS detector, respectively. Aqueous samples, containing iron salts and other inorganic material, for GC analyses were extracted with several portions of dichloromethane and concentrated under reduced pressure.
TOC analyses were performed on Shimadzu TOC5000A Total organic carbon analyzer (Shimadzu, Kyoto, Japan). Confirmation of structures of independently synthesysed products, HRMS measurements were performed on Agilent 6224 TOF LC/MS system (Agilent, Santa Clara, CA, USA), and NMR spectra were measured on Bruker Avance III 500 spectrometer (Bruker, Karlsruhe, Germany).

Wang Z., Yao J., Bavcon Kralj M., Dolenc D, & Trebše P. (2021). Removal of Flotation Collector O-Isopropyl-N-ethylthionocarbamate from Wastewater. Molecules, 26(21), 6676.

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Agilent 6224 tof lc ms system

Lab products found in correlation

5 protocols using agilent 6224 tof lc ms system

Enzymatic Characterization of GlcNAc Oligosaccharide

CE-TOFMS-based Metabolome Profiling

Metabolomic Analysis of Stable Isotope Labeling in Cell Lines

Metabolomic Analysis of Stable Isotope Labeling in Cell Lines

Analytical Techniques for Reaction Mixture

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