Agilent ce system

Manufactured by Agilent Technologies

Sourced in United States

The Agilent CE system is a capillary electrophoresis platform designed for the separation and analysis of a wide range of biomolecules, including proteins, nucleic acids, and small molecules. It utilizes the principles of electrophoresis to separate analytes based on their charge-to-size ratio, allowing for high-resolution separation and detection.

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16 protocols using agilent ce system

Quantitative Metabolomic Analysis by CE-MS

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Quantitative metabolome analysis was performed using CE-MS27 (link). Briefly, to extract metabolites from the tissue, the frozen tissue block embedded in SCEM medium together with internal control compounds (see below) was homogenized in ice-cold methanol (500 μL) using a manual homogenizer (Finger Masher (AM79330); Sarstedt, Tokyo, Japan), followed by the addition of an equal volume of chloroform and 0.4 times the volume of ultrapure water (LC/MS grade; Wako). The suspension was then centrifuged at 15,000 g for 15 min at 4 °C. After centrifugation, the aqueous phase was ultrafiltered using an ultrafiltration tube (Ultrafree-MC, UFC3 LCC NB; Human Metabolome Technologies, Tsuruoka, Japan). The filtrate was concentrated with a vacuum concentrator (SpeedVac; Thermo, Yokohama, Japan); this condensation process helps quantitate trace levels of metabolites. The concentrated filtrate was dissolved in 50 μL of ultrapure water and used for CE-MS.
All CE-MS experiments were performed using an Agilent CE System equipped with an air pressure pump, an Agilent 6520 Accurate Q-Tof mass spectrometer, an Agilent 1200 series isocratic high-performance LC pump, 7100 CE-system, a G1603A Agilent CE-MS adapter kit, and a G1607A Agilent CE-MS sprayer kit (Agilent Technologies). For more detail, see the supplementary information.

Sugiura Y., Katsumata Y., Sano M., Honda K., Kajimura M., Fukuda K, & Suematsu M. (2016). Visualization of in vivo metabolic flows reveals accelerated utilization of glucose and lactate in penumbra of ischemic heart. Scientific Reports, 6, 32361.

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Fecal Metabolome Analysis by CE-TOFMS

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Capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS)-based metabolome analysis of fecal samples was conducted as described previously (Hirayama et al., 2012 (link)) with some modifications. In brief, fecal samples were lyophilized using a VD-800R lyophilizer (TAITEC) for 24 h. Freeze-dried feces were disrupted with 3.0-mm Zirconia Beads (Biomedical Science) by vigorous shaking (1,500 rpm for 10 min) using the Shake Master (Biomedical Science). Fecal metabolites were extracted using the methanol:chloroform:water extraction protocol (Wang et al., 2015 (link)). CE-TOFMS experiments were performed using the Agilent CE System, the Agilent G3250AA LC/MSD TOF System, the Agilent 1100 Series Binary HPLC Pump, the G1603A Agilent CE-MS adapter, and the G1607A Agilent CE-ESI-MS Sprayer kit. In-house software (MasterHands) (Sugimoto et al., 2010 (link)) was used for data processing, quantification, and peak annotation.

Tanaka K., Tanigawa N., Song I., Komatsu T., Kuriki Y., Tanaka Y., Fukudo S., Urano Y, & Fukuda S. (2023). A protease activity-based machine-learning approach as a complementary tool for conventional diagnosis of diarrhea-predominant irritable bowel syndrome. Frontiers in Microbiology, 14, 1179534.

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Fecal Metabolome Analysis via CE-TOFMS

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Capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS)–based metabolome analysis was conducted as described previously with some modifications.²⁸ (link) In brief, fecal samples were lyophilized by using a VD-800R lyophilizer (TAITEC, Saitama, Japan) for 24 hours. Freeze-dried feces were disrupted with 3.0-mm Zirconia Beads (Biomedical Science, Tokyo, Japan) by vigorous shaking (1500 rpm for 10 min) using Shake Master (Biomedical Science). Fecal metabolites were extracted by the methanol:chloroform:water extraction protocol. CE-TOFMS experiments were performed using the Agilent CE System, the Agilent G3250AA LC/MSD TOF System, the Agilent 1100 Series Binary HPLC Pump, the G1603A Agilent CE-MS adapter, and the G1607A Agilent CE-ESI-MS SprayerKit (all Agilent Technologies, Santa Clara, CA).

Nagao-Kitamoto H., Shreiner A.B., Gillilland MG I.I.I., Kitamoto S., Ishii C., Hirayama A., Kuffa P., El-Zaatari M., Grasberger H., Seekatz A.M., Higgins P.D., Young V.B., Fukuda S., Kao J.Y, & Kamada N. (2016). Functional Characterization of Inflammatory Bowel Disease–Associated Gut Dysbiosis in Gnotobiotic Mice. Cellular and Molecular Gastroenterology and Hepatology, 2(4), 468-481.

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Fecal Metabolome Analysis by CE-TOFMS

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Capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS)–based metabolome analysis was conducted as described previously with some modifications ^{44 (link)}. In brief, fecal samples were lyophilized using a VD-800R lyophilizer (TAITEC) for 24 hours. Freeze-dried feces were disrupted with 3.0-mm Zirconia Beads (Biomedical Science) by vigorous shaking (1,500 rpm for 10 min) using ShakeMaster (Biomedical Science). Fecal metabolites were extracted using the methanol:chloroform:water extraction protocol. CE-TOFMS experiments were performed using the Agilent CE System, the Agilent G3250AA LC/MSD TOF System, the Agilent 1100 Series Binary HPLC Pump, the G1603A Agilent CE-MS adapter and the G1607A Agilent CE-ESI-MS Sprayer Kit (Agilent Technologies). MasterHands software was used for data processing, quantification and peak annotation ^{45 (link)}.

Kitamoto S., Alteri C.J., Rodrigues M., Nagao-Kitamoto H., Sugihara K., Himpsl S.D., Bazzi M., Miyoshi M., Nishioka T., Hayashi A., Morhardt T.L., Kuffa P., Grasberger H., El-Zaatari M., Bishu S., Ishii C., Hirayama A., Eaton K.A., Dogan B., Simpson K.W., Inohara N., Mobley H.L., Kao J.Y., Fukuda S., Barnich N, & Kamada N. (2019). Dietary L-serine confers a competitive fitness advantage to Enterobacteriaceae in the inflamed gut. Nature microbiology, 5(1), 116-125.

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Metabolite Extraction and Analysis by CE-TOFMS

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The cecum luminal contents were immediately frozen in liquid nitrogen and stored at −80 °C until metabolite extraction. Sample tissues were weighed and completely homogenized in 0.5 mL of ice-cold methanol containing 50 μM methionine sulfone and camphor-10-sulfonic acid as internal standards. The homogenates were mixed with 0.5 mL of chloroform and 0.2 mL of ice-cold Milli-Q water. After centrifugation at 2300× g for 5 min, the supernatant was centrifugally filtrated through 5 kDa cut-off filters (Millipore, Bedford, MA, USA) at 9100× g for 4 to 5 h to remove proteins. The filtrate was then centrifugally concentrated in a vacuum evaporator, dissolved in Milli-Q water, and analyzed by capillary electrophoresis electrospray ionization time-of-flight mass spectrometry (CE-TOFMS) using an Agilent CE system combined with a TOFMS (Agilent Technologies, Palo Alto, CA, USA) as reported by Human Metabolome Technologies, Inc. (HMT) (HMT, Tsuruoka, Japan) [21 (link),22 (link)]. Each metabolite was identified and quantified based on the peak information, including m/z, migration time, and peak area.

Uebanso T., Ohnishi A., Kitayama R., Yoshimoto A., Nakahashi M., Shimohata T., Mawatari K, & Takahashi A. (2017). Effects of Low-Dose Non-Caloric Sweetener Consumption on Gut Microbiota in Mice. Nutrients, 9(6), 560.

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Plasma Metabolome Profiling by CE-TOFMS

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Plasma (100 μl each) was added to 0.9 ml of ice-cold methanol containing 10 μM methionine sulfone as an internal standard. The mixtures were vortexed with 1 ml of chloroform and 0.4 ml of ice-cold Milli-Q water. After centrifugation at 2,300 × g for 5 min at 4°C, the supernatant was centrifugally filtrated through 5-kDa cut-off filters (Millipore, Bedford, MA) at 9,100 × g for 4 to 5 h to remove proteins. The filtrate was centrifugally concentrated in a vacuum evaporator, dissolved in Milli-Q water, and analyzed by capillary electrophoresis electrospray ionization time-of-flight mass spectrometry (CE-TOFMS).
The CE-TOFMS analysis was performed using an Agilent CE system combined with a TOFMS (Agilent Technologies, Palo Alto, CA). Each metabolite was identified in reference to internal standards (H3304-1002, Human Metabolome Technology, Inc., Tsuruoka, Japan) for m/z and migration time, and quantified by peak area.

Yano Y., Maeda C., Kaneko I., Kobayashi Y., Aoi W, & Kuwahata M. (2021). Cystine supplementation sustains plasma mercaptalbumin levels in rats fed low-protein diets more effectively than methionine. Journal of Clinical Biochemistry and Nutrition, 69(2), 122-130.

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Metabolomic Analysis by CE-TOFMS and LC-TOFMS

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Metabolome analysis was conducted using a Dual Scan package at HMT using CE-TOFMS and LC-TOFMS for ionic and non-ionic metabolites, respectively, based on previously described methods [15 (link),16 (link)]. The CE-TOFMS analysis were carried out using an Agilent CE system (Agilent Technologies, Palo Alto, CA, USA) equipped with an Agilent 6210 TOFMS (Agilent Technologies) at a service facility at HMT. Cationic metabolites were analyzed with a fused silica capillary (50 μm internal diameter × 80 cm total length) with cationic electrophoresis buffer (H3301-1001, HMT) as the electrolyte. The sample solution was injected at a pressure of 50 mbar for 10 s. The applied voltage was set at 27 kV. Electrospray ionization-mass spectrometry (ESI-MS) was conducted in the positive-ion mode and the capillary voltages were set at 4000 V. The spectrometer was scanned from m/z 50 to 1000. Anionic metabolites were analyzed with a fused silica capillary (50 μm × 80 cm) with anionic electrophoresis buffer (H3302-1021, HMT) as the electrolyte. The sample solution was injected at a pressure of 50 mbar for 25 s. The applied voltage was set at 30 kV. ESI-MS was conducted in the negative-ion mode and the capillary voltages were set at 3500 V. The spectrometer was scanned from m/z 50 to 1000.

Hiroshima Y., Yamamoto T., Watanabe M., Baba Y, & Shinohara Y. (2018). Effects of cold exposure on metabolites in brown adipose tissue of rats. Molecular Genetics and Metabolism Reports, 15, 36-42.

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CE-MS Metabolite Identification and Quantification

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The CE-MS analysis was performed using an Agilent CE system equipped with a time-of-flight mass spectrometer (TOF-MS) (Agilent Technologies, USA) as previously described [54 (link),55 (link),56 (link),57 (link)]. Metabolites in the samples were identified by comparing the migration times (MTs) and m/z ratios with those of authentic standards. The tolerance was ±0.5 min for MTs and ±10 ppm for the m/z ratios. Metabolites in the samples were quantified by comparing their peak areas with those of the authentic standards using ChemStation software (Agilent Technologies). Data analyses were conducted using GraphPad Prism. The variance of sample data was analysed by ANOVA using Statistical Analysis Software (SAS) version 9.4 (SAS Inc., Cary, NC, USA).

Ali M.S, & Baek K.H. (2020). Co-Suppression of NbClpC1 and NbClpC2, Encoding Clp Protease Chaperons, Elicits Significant Changes in the Metabolic Profile of Nicotiana benthamiana. Plants, 9(2), 259.

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Metabolite Profiling of Liver, Muscle, and Plasma

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Metabolite measurements for this study were performed on the liver, skeletal muscle and plasma using CE-MS and LC-MS. CE-MS was performed using an Agilent CE system coupled to an Agilent 6520 Q-TOF (Agilent Technologies). LC-MS was performed using an Agilent 1260 Series coupled to an Agilent 6520 Q-TOF. The HPLC system was equipped with a CAPCELL PAK C18 IF column (2·0 mm I.D. × 50 mm, 2 μm) or a Poroshell 120 EC-C18 (2·1 mm I.D. × 150 mm, 2·7 μm). For each metabolite measured by metabolome analysis, the relative peak area was calculated and the values were listed.

Furuya F., Fujita Y., Matsuo N., Minamino H., Oguri Y., Isomura N., Ikeda K., Takesue K., Li Y., Kondo A., Mano F, & Inagaki N. (2022). Liver autophagy-induced valine and leucine in plasma reflect the metabolic effect of sodium glucose co-transporter 2 inhibitor dapagliflozin. eBioMedicine, 86, 104342.

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Metabolite Profiling of Resected Liver Tissues

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Resected liver samples were immediately frozen and stored at −80°C until metabolite extraction. Sample tissues were weighed and completely homogenized by the Multi-Beads Shocker (Yasuikikai). Homogenates were mixed with 0.5-mL chloroform and 0.2-mL ice-cold Milli-Q water. After centrifugation at 2300 g for 5 minutes, the supernatant was centrifugally filtrated through 5-kDa cut-off filters (Milli-pore) to remove proteins. Filtrate was centrifugally concentrated in a vacuum evaporator, dissolved with Milli-Q water and analysed by CE-TOFMS. CE-TOFMS analysis was performed by the Agilent CE System combined with a TOFMS (Agilent Technologies, Santa Clara, CA, USA), as described previously, ^{19 (link)} with slight modifications. Each metabolite was identified and quantified based on the peak information including m/z (mass-to- charge ratio), migration time and peak area. A total of 116 metabolites involved in glycogen synthesis, the pentose phosphate pathway, glycolysis, the TCA cycle, the urea cycle, and metabolism of polyamine, creatine, purine, glutathione, nicotinamide, choline and amino acid were quantified (Tables S2 and S3). Quantified data were evaluated for statistical significance by the Wilcoxon signed-rank test.

Alves-Paiva R.M., Kajigaya S., Feng X., Chen J., Desierto M., Wong S., Townsley D.M., Donaires F.S., Bertola A., Gao B., Young N.S, & Calado R.T. (2017). Telomerase enzyme deficiency promotes metabolic dysfunction in murine hepatocytes upon dietary stress. Liver international : official journal of the International Association for the Study of the Liver, 38(1), 144-154.

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