Cells from 10 cm dishes were washed twice with 10 ml 5% mannitol before metabolites were extracted from melanoma cell pellets using 1 mL methanol for 10 min and samples were deproteinized using 400 μl CHCl3 and 200 ml MilliQ water followed by centrifugation at 10,000 g for 3 min at 4°C. 400 μl of the aqueous layer was then filtered using a 5 kDa ultrafiltration tube and analysis by capillary electrophoresis mass spectrometry (CE‐MS) after addition of 25 μl 200 mM internal standards: L-Methionine sulfone (Wako 502–76641), 2-(N-morpholino) ethanesulfonic acid (Dojindo 349–01623) and D-Camphor-10- sulfonic Acid (Wako 037–01032); 3-Aminopyrrolidine (Aldrich 404624) and Trimesate (Wako 206–03641) as described (Kami et al., 2013 (link)) on an Agilent capillary electrophoresis system consisting of an Agilent G6220A LC/MSD TOF, an Agilent 1100 series isocratic HPLC pump, a G1603A Agilent CE-MS adapter kit, and a G1607A Agilent CE-ESI-MS sprayer kit (Agilent Technologies, USA).
2 n morpholino ethanesulfonic acid
Manufactured by Dojindo Laboratories
Sourced in United States, Japan
2-(N-morpholino) ethanesulfonic acid is a chemical compound used as a buffer in various biochemical applications. It maintains a stable pH environment for biological processes.
Automatically generated - may contain errors
Lab products found in correlation
D camphor 10 sulfonic acid, by Fujifilm (3 mentions)
3 aminopyrrolidine, by Merck Group (3 mentions)
Trimesate, by Fujifilm (2 mentions)
L methionine sulfone, by Fujifilm (2 mentions)
G6220a lc msd tof, by Agilent Technologies (2 mentions)
1100 series isocratic hplc pump, by Agilent Technologies (2 mentions)
Agilent g1607a ce esi ms sprayer kit, by Agilent Technologies (2 mentions)
Agilent g1603a ce ms adapter kit, by Agilent Technologies (2 mentions)
Trimesic acid, by Fujifilm (1 mentions)
Agilent ce tofms system, by Agilent Technologies (1 mentions)
Methionine sulfone, by Fujifilm (1 mentions)
3 mm zirconia beads, by Biospec (1 mentions)
Sucrose monolaurate, by Merck Group (1 mentions)
Tetramethylsilane, by Cambridge Isotopes (1 mentions)
4 protocols using 2 n morpholino ethanesulfonic acid
1
Metabolite Extraction and Analysis of Melanoma Cells
2
Quantitative Metabolite Analysis of Quadriceps
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A quantitative analysis of charged metabolites in the quadriceps femoris was performed using CE-TOFMS as described previously (66 (link), 67 (link)). Quadriceps femoris muscle samples (50 mg) were submerged in methanol (500 μl) containing internal standards [methionine sulfone (Wako, Tokyo, Japan), 2-(N-morpholino)ethane sulfonic acid (Dojindo Laboratories, Tokyo, Japan), and d -camphor-10-sulfonic acid (Wako) each at 20 μM]. The mixture was combined with four 3-mm zirconia beads (BioSpec Products, Bartlesville, OK, USA) and vigorously shaken for 5 min using a Shake Master NEO (Biomedical Science, Tokyo, Japan). Subsequently, deionized water (200 μl) and chloroform (500 μl) were added, and the suspension was vigorously shaken for 5 min and centrifuged at 4600g and 4°C for 30 min. The supernatant was filtered through a 5-kDa-cutoff filter column (Millipore, Burlington, MA, USA). The filtrate was concentrated via centrifugation (3 hours at 40°C) and dissolved in water containing reference compounds [200 μM each of 3-aminopyrrolidine (Sigma-Aldrich, St. Louis, MO, USA) and trimesic acid (Wako)] before CE-TOFMS analysis. As described previously (68 (link)), the CE-TOFMS experiments were performed using an Agilent CE-TOFMS system (Agilent Technologies).
3
Melanoma Metabolite Extraction and Analysis
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Cells from 10 cm dishes were washed twice with 10 ml 5% mannitol in MilliQ water before metabolites were extracted from melanoma cell pellets using 1 ml methanol for 10 min and samples were deproteinized using 400 µl CHCl3 and 200 ml MilliQ water followed by centrifugation at 10,000 g for 3 min at 4°C. 400 µl of the aqueous layer was then filtered using a 5 kDa ultrafiltration tube and analysed by capillary electrophoresis mass spectrometry (CE‐MS) after addition of 25 µl 200 mM internal standards: L‐methionine sulfone (Wako 502–76641), 2‐(N‐morpholino) ethanesulfonic acid (Dojindo 349–01623), D‐Camphor‐10‐sulfonic acid (Wako 037–01032), 3‐aminopyrrolidine (Aldrich 404624) and trimesate (Wako 206–03641) as described (Kami et al., 2013 ) on an Agilent capillary electrophoresis system consisting of an Agilent G6220A LC/MSD TOF, an Agilent 1100 series isocratic HPLC pump, a G1603A Agilent CE‐MS adapter kit, and a G1607A Agilent CE‐ESI‐MS sprayer kit (Agilent Technologies, USA).
4
Enzymatic Production of D-Glyceric Acid
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D-Glyceric acid was prepared by the oxidative fermentation of glycerol by Acetobacter tropicalis NBRC14670, according to the method reported by Habe et al. 4) Sucrose phosphorylase was obtained from Oriental Yeast (Osaka, Japan) . N-Dodecylamine, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride n -hydrate (DMT-MM) was purchased from Wako Pure Chemicals (Osaka, Japan) . Sucrose monolaurate was obtained from Sigma-Aldrich (St. Louis, MO, USA) . 2-(N-Morpholino) ethanesulfonic acid and MEGA-10 (n-decanoyl-N-methyl-Dglucamine) were obtained from Dojindo Laboratories (Kumamoto, Japan) . Methanol-d 4 (d 99.8％) plus 0.05％ (v/v) tetramethylsilane was obtained from Cambridge Isotope Laboratories (Tewksbury, MA, USA) . Other reagents and solvents were of analytical grade and were obtained from Wako Pure Chemicals and Tokyo Chemical Industries (Tokyo, Japan) .
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