Ce capillary electrophoresis system

Manufactured by Agilent Technologies

Sourced in Germany

The CE capillary electrophoresis system is a laboratory instrument used for the separation and analysis of a wide range of molecules, including proteins, nucleic acids, and small molecules. The system utilizes a capillary filled with a conductive buffer and an applied electric field to separate the sample components based on their electrophoretic mobility.

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8 protocols using ce capillary electrophoresis system

Metabolic Profiling of Gut Microbiome

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Small intestine, cecal and colonic contents from mice were collected, immediately frozen using dry ice and stored at −80°C until use. 10 mg of freeze-dried small intestine, cecal or colonic luminal samples were mixed with 500 μl methanol containing internal standards: 20 μM each of methionine sulfone and D-camphor-10-sulfonic acid. Samples were homogenized in a Shake Master NEO machine (Bio Medical Science Inc.) using 0.1 mm zirconia and silica beads (BioSpec). 200 μl of Milli-Q water and 500 μl of chloroform were added to the mixtures and samples were shaken again. Samples were centrifuged (4,600×g/15min, 4°C), supernatants were concentrated at 40°C using a 5 kDa cutoff centrifugal filter and pellets reconstituted with 40 μl of Milli-Q water. Metabolites were analyzed using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) in both positive and negative modes using a CE capillary electrophoresis system (Agilent Technologies). Peak annotation and quantification were performed using an in-house software (MasterHands) (Sugimoto et al., 2010 (link); Yamamoto et al., 2018 ). AA amounts were visualized as heat-maps using MeV (http://mev.tm4.org/).

Caballero-Flores G., Pickard J.M., Fukuda S., Inohara N, & Núñez G. (2020). An enteric pathogen subverts colonization resistance by evading competition for amino acids in the gut. Cell host & microbe, 28(4), 526-533.e5.

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Metabolome Profiling by CE-TOFMS

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CE-TOFMS was performed on an Agilent CE Capillary Electrophoresis System equipped with an Agilent 6210 TOF mass spectrometer, Agilent 1100 isocratic HPLC pump, Agilent G1603A CE-MS adapter kit, and Agilent G1607A CE-ESI-MS sprayer kit (Agilent Technologies, Waldbronn, Germany). Metabolome measurements were performed at Human Metabolome Technologies as previously described (Soga and Heiger 2000 (link); Soga et al. 2002 (link); Soga et al. 2003 (link)). For details of the measurement, see supporting information.

Tani T., Okamoto K., Fujiwara M., Katayama A, & Tsuruoka S. (2019). Metabolomics analysis elucidates unique influences on purine / pyrimidine metabolism by xanthine oxidoreductase inhibitors in a rat model of renal ischemia-reperfusion injury. Molecular Medicine, 25, 40.

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Capillary Electrophoresis-Time of Flight Mass Spectrometry

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Capillary electrophoresis-time of flight mass spectrometry (CE-TOFMS) was performed using an Agilent CE capillary electrophoresis system equipped with an Agilent 6210 time of flight mass spectrometer, Agilent 1100 isocratic high-performance liquid chromatography (HPLC) pump, Agilent G1603A CE-MS adapter kit, and Agilent G1607A CE-electrospray ionization (ESI)-MS sprayer kit (Agilent Technologies, Waldbronn, Germany). The system was controlled by Agilent G2201AA ChemStation software for CE. Data acquisition was performed by Analyst QS software for Agilent TOF (Applied Biosystems, CA; MDS Sciex, Ontario, Canada).

Jeelani G., Sato D., Soga T., Watanabe H, & Nozaki T. (2014). Mass Spectrometric Analysis of l-Cysteine Metabolism: Physiological Role and Fate of l-Cysteine in the Enteric Protozoan Parasite Entamoeba histolytica. mBio, 5(6), e01995-14.

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Capillary Electrophoresis-Mass Spectrometry

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Capillary electrophoresis–mass spectrometry was performed as previously described (Kinoshita et al., 2007 (link); Yamamoto et al., 2014 (link)). The instrument used was an Agilent CE Capillary Electrophoresis System equipped with an air pressure pump, an Agilent 1100 series isocratic high-performance liquid chromatography pump and an Agilent 1100 series MSD mass spectrometer. We also used a G1603A Agilent CE-MS adapter kit, and a G1607A Agilent CE-MS sprayer kit (Agilent Technologies). G2201AA Agilent ChemStation software for CE-MSD was used for system control and data acquisition.

Nurkanto A., Jeelani G., Yamamoto T., Hishiki T., Naito Y., Suematsu M., Hashimoto T, & Nozaki T. (2018). Biochemical, Metabolomic, and Genetic Analyses of Dephospho Coenzyme A Kinase Involved in Coenzyme A Biosynthesis in the Human Enteric Parasite Entamoeba histolytica. Frontiers in Microbiology, 9, 2902.

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Synchronized Synechococcus Metabolite Analysis

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Synchronized Synechococcus 7942 wild-type cells were harvested with fast filtration and immediately suspended in the extraction solution, as described previously [29 (link)]. Metabolite concentrations were analysed with capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), using an Agilent CE capillary electrophoresis system equipped with an Agilent 6210 time-of-flight mass spectrometer, an Agilent 1100 isocratic HPLC pump, an Agilent G1603A CE-MS adapter kit, and an Agilent G1607A CE-ESI-MS sprayer kit with an attached platinum needle (Agilent Technologies, Waldbronn, Germany). Cationic metabolites were separated with a fused silica capillary, using 1 M formic acid as the electrolyte. Anionic metabolites and nucleotides were separated with a COSMO (+) capillary (Nacalai Tesque, Kyoto, Japan), using 50 mM ammonium acetate (pH 8.5) as the electrolyte. Details of the CE-TOFMS conditions and the methods used for data analysis were described previously [30 (link)]. Absolute quantifications were performed using metabolite standards and calculated as the amount of metabolite per 10⁷ cells (i.e. pmol per 1 × 10⁷ cells) in each sample (S2 Table). We were unable to separate the TOF-MS peaks of 3-phosphoglycerate (3-PG) and 2-phosphoglycerate (2-PG). Consequently, they are depicted as the combined total amounts of 3-PG and 2-PG.

Watanabe S., Ohbayashi R., Kanesaki Y., Saito N., Chibazakura T., Soga T, & Yoshikawa H. (2015). Intensive DNA Replication and Metabolism during the Lag Phase in Cyanobacteria. PLoS ONE, 10(9), e0136800.

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Metabolome Profiling by CE-TOFMS

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Metabolomes were profiled by CE-TOFMS using an Agilent CE Capillary Electrophoresis System equipped with a 6210 Time-of-Flight mass spectrometer, an 1100 isocratic HPLC pump, a G1603A CE-MS adapter kit and a G1607A CE-ESI-MS sprayer kit (all from Agilent Technologies, Waldbronn, Germany), and this system was controlled by G2201AA ChemStation software version B.03.01 for CE (Agilent Technologies).

Matsuura Y., Yamashita A., Zhao Y., Iwakiri T., Yamasaki K., Sugita C., Koshimoto C., Kitamura K., Kawai K., Tamaki N., Zhao S., Kuge Y, & Asada Y. (2017). Altered glucose metabolism and hypoxic response in alloxan-induced diabetic atherosclerosis in rabbits. PLoS ONE, 12(4), e0175976.

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Intracellular Metabolite Profiling of Activated T Cells

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CD3⁺ T cells (around 1.8 × 10⁷ cells per sample) were activated for 36 hours in conditional media (RPMI-1640) containing 2 mM ¹³C₅-glutamine, 1.15 mM ¹³C₆-arginine, or 0.17 mM ¹³C₅-proline, and then used for the extraction of intracellular metabolites as described above. Metabolome measurements were carried out through a facility service at Human Metabolome Technologies Inc. (Tsuruoka, Japan). CE-TOFMS measurement was carried out using an Agilent CE Capillary Electrophoresis System equipped with an Agilent 6210 Time of Flight mass spectrometer, Agilent 1100 isocratic HPLC pump, Agilent G1603A CE-MS adapter kit, and Agilent G1607A CE-ESI-MS sprayer kit (Agilent Technologies, Waldbronn, Germany). The systems were controlled by Agilent G2201AA ChemStation software version B.03.01 for CE (Agilent Technologies, Waldbronn, Germany). The metabolites were analyzed by using a fused silica capillary [50 μm internal diameter (i.d.)× 80 cm total length], with commercial electrophoresis buffer (solution ID: H3301-1001 for cation analysis and H3302-1021 for anion analysis, Human Metabolome Technologies) as the electrolyte. The sample was injected at a pressure of 50 mbar for 10 s (approximately 10 nl) in the cation analysis and 25 s (approximately 25 nl) in the anion analysis. The spectrometer was scanned from m/z 50 to 1000.

Wu R., Chen X., Kang S., Wang T., Gnanaprakasam J.R., Yao Y., Liu L., Fan G., Burns M.R, & Wang R. (2020). De novo synthesis and salvage pathway coordinately regulate polyamine homeostasis and determine T cell proliferation and function. Science Advances, 6(51), eabc4275.

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High-Performance Liquid Chromatography and Micellar Electrokinetic Chromatography Protocols

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HPLC measurements were done using a 10 A series chromatograph from Shimadzu (Kyoto, Japan) equipped with a quaternary pump and a diode array detector and fitted with either an IAM.DD 2 immobilized artificial membrane column (10 cm × 4.6 mm i.d., 12 m particle size) (Regis Technologies, Morton Grove, IL, US) or a XBridge C18 column (15 cm × 4.6 mm i.d., 5 m particle size) (Waters, Milford, MA, US). MEKC measurements were done using the CE capillary electrophoresis system from Agilent Technologies (Santa Clara, CA, US) equipped with a diode array detector. The fused-silica separation capillary (40 cm effective length, 50 μm i.d.) was obtained from Composite Metal Services Ltd (Shipley, UK).

Fernández-Pumarega A., Amézqueta S., Fuguet E, & Rosés M. (2015). Tadpole toxicity prediction using chromatographic systems. Journal of chromatography. A, 1418.

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