Extracts were resuspended in 5% ACN with 0.2% formic acid to a final concentration of 2 μg/μL. For LC-MS analysis, 40 μg of sample was loaded onto a 150 mm × 2.1 mm i.d., 2 μm particle size Kinetex C18 RPLC column (Phenomenex, Torrance, CA). Analysis was performed using an Agilent 1150 LC system (Agilent, Santa Clara, CA) equipped with a photodiode array and placed in-line with a Q-Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Chromatography was performed at a flow rate of 200 μL/min using water/0.1% formic acid (solvent A) and acetonitrile/0.1% formic acid (solvent B) with the following gradient: time 0 min., 2% B; 35 min., 60% B; 54 min., 98% B. UV spectra were acquired at a rate of 1 Hz. For every spectrum, the three most intense apices were recorded. The mass spectrometer instrument settings were as follows: capillary temperature 275 °C, sheath gas 8 (arbitrary units), spray voltage 4.2 kV. Full MS spectra were acquired at 35,000 resolution for the mass range m/z 250 to 3750 for all samples. This resulted in an average scan rate of 6 Hz. Following each full MS scan, the top 5 most intense ions were selected for a dependent MS2 scan. MS2 was conducted using HCD with a collisional energy of 25%.
Agilent 1150 lc system
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 1150 LC system is a liquid chromatography (LC) system designed for analytical and preparative separation and purification of chemical compounds. It features a quaternary pump, an autosampler, a column compartment, and a UV-Vis detector. The system is capable of performing gradient and isocratic elution.
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4 protocols using agilent 1150 lc system
1
LC-MS Analysis of Protein Extracts
2
LC-MS Analysis of Protein Extracts
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Extracts were resuspended in 5% ACN with 0.2% formic acid to a final concentration of 2 μg/μL. For LC-MS analysis, 40 μg of sample was loaded onto a 150 mm × 2.1 mm i.d., 2 μm particle size Kinetex C18 RPLC column (Phenomenex, Torrance, CA). Analysis was performed using an Agilent 1150 LC system (Agilent, Santa Clara, CA) equipped with a photodiode array and placed in-line with a Q-Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Chromatography was performed at a flow rate of 200 μL/min using water/0.1% formic acid (solvent A) and acetonitrile/0.1% formic acid (solvent B) with the following gradient: time 0 min., 2% B; 35 min., 60% B; 54 min., 98% B. UV spectra were acquired at a rate of 1 Hz. For every spectrum, the three most intense apices were recorded. The mass spectrometer instrument settings were as follows: capillary temperature 275 °C, sheath gas 8 (arbitrary units), spray voltage 4.2 kV. Full MS spectra were acquired at 35,000 resolution for the mass range m/z 250 to 3750 for all samples. This resulted in an average scan rate of 6 Hz. Following each full MS scan, the top 5 most intense ions were selected for a dependent MS2 scan. MS2 was conducted using HCD with a collisional energy of 25%.
3
Metabolomics of Inactivated GABA-AT
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Example 28
Metabolomics of the Inactitaved GABA-AT. A 50 μL portion of ammonium bicarbonate buffer (50 mM, pH 7.4) containing GABA-AT (23 μg, 0.21 nmol), α-ketoglutarate (5 mM), and 17 (43 mM) was protected from light and incubated at rt overnight until the activity of GABA-AT was less than 1%. Another 50 μL of ammonium bicarbonate buffer (50 mM, pH 7.4) containing GABA-AT (23 μg, 0.21 nmol) and α-ketoglutarate (5 mM) was subjected to the same condition as a control. After incubation, formic acid (10 μL) was added to each sample, and 20 μL of each resulting solution was loaded onto a 5-μm Luna C18 column (2 mm i.d.; 150 mm) (Phenomenex, Torrance, Calif., USA). A 30-min LC gradient was employed at a flow rate of 200 μl/min on an Agilent 1150 LC system (Agilent, Santa Clara, Calif., USA). Mass spectrometry was performed on a Q-Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, Mass., USA). Intact MS spectra were acquired at a resolution of 35,000. The top-five most intense ions were selected for fragmentation in a data-dependent acquisition mode. Mass spectra were acquired at a resolution of 17,500.
4
Metabolomic Profiling of Inactivated GABA-AT
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Example 28
Metabolomics of the Inactitaved GABA-AT.
A 50 μL portion of ammonium bicarbonate buffer (50 mM, pH 7.4) containing GABA-AT (23 μg, 0.21 nmol), α-ketoglutarate (5 mM), and 17 (43 mM) was protected from light and incubated at rt overnight until the activity of GABA-AT was less than 1%. Another 50 ∞L of ammonium bicarbonate buffer (50 mM, pH 7.4) containing GABA-AT (23 μg, 0.21 nmol) and α-ketoglutarate (5 mM) was subjected to the same condition as a control. After incubation, formic acid (10 μL) was added to each sample, and 20 μL of each resulting solution was loaded onto a 5-μm Luna C18 column (2 mm i.d.; 150 mm) (Phenomenex, Torrance, Calif., USA). A 30-min LC gradient was employed at a flow rate of 200 μl/min on an Agilent 1150 LC system (Agilent, Santa Clara, Calif., USA). Mass spectrometry was performed on a Q-Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, Mass., USA). Intact MS spectra were acquired at a resolution of 35,000. The top-five most intense ions were selected for fragmentation in a data-dependent acquisition mode. Mass spectra were acquired at a resolution of 17,500.
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