Initial optimization with angiotensin was studied using a Waters Acquity ultrahigh-performance liquid chromatography (UPLC) (Empower Software) connected to an Advion Expression CMS mass spectrometer. Method: Solvent A: acetonitrile, Solvent B: water, (5% A and 95% B, 0–2 min; 10% A and 90%, 2–3 min; 20% A and 80% B, 3–10 min; 30% A and 70% B, 10–12 min; 95% A and 5% B, 12–16.5 min; 95% A and 5% B, 16.5–18 min; 5% A and 95% B, 18–20 min). For the further optimization of angiotensin at UT-Austin, LC/MS were recorded on an Agilent Technologies 6120 Single Quadrupole or 6130 Single Quadrupole interfaced with an Agilent 1200 series LC system equipped with a diode-array detector. The resulting spectra were analyzed using Agilent LC/MSD ChemStation. All LC experiments were run with a 5–95% gradient elution (methanol/water) over 15 min. The conversion was calculated by the ratio of the peak areas of C-terminal-modified angiotensin and p-toluic acid (internal standard) under UV trace for angiotensin photoredox reaction optimization reaction (Supporting Information Table 1–4 ) For the determination of conversions of peptides with varying C-terminal AAs (Table 1 ), the value was obtained by dividing the peak area of the C-terminal modified product by the addition of peak areas of the C-terminal modified product and unreacted starting materials under the total ion chromatogram (TIC).
Agilent lc msd chemstation
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The Agilent LC/MSD ChemStation is a software platform that provides instrument control, data acquisition, and data analysis for Agilent liquid chromatography and mass spectrometry systems. It enables users to perform routine analysis and complex method development.
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3 protocols using agilent lc msd chemstation
1
Optimization of Angiotensin Photoredox Reaction
2
Kinetic Binding Assay of MN551
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SBC2 protein (40 µM), SBC2 C111S, SBC4, SBC6, or CISH were incubated with an equimolar concentration of MN551 in 50 mM Tris, pH 8.0, 50 mM NaCl, 1 mM TCEP. At defined time points 20 µl samples were removed and the protein precipitated by the addition of 80 µl methanol. The precipitated protein was pelleted by centrifugation, washed with 500 µl methanol, and resuspended in an aqueous solution of 15% acetonitrile and 1% TFA. Samples were separated by HPLC on a C3 column using a 10–75% gradient of acetonitrile and analyzed using an Agilent 6130 quadrupole MS. Spectra were deconvoluted and integrated using Agilent LC/MSD ChemStation.
3
Quantitative LC-MS Analysis of NRGs
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LC-MS analysis was performed using an Agilent 1100 HPLC system, consisting of a G1311A quaternary pump and solvent module, a G1322A vacuum degasser, a G1313A autosampler (ALS), a G1315A diode-array (DAD) detector (operating at 280 nm) and a G1316A thermostatted column compartment (TCC, kept at a constant temperature of 25 °C). The HPLC system was equipped with a Grace Prevail reversed-phase C18 3μm column (length: 150 mm, ID: 2.1 mm) and coupled to an Agilent 6110 single-quadrupole mass spectrometer with an electrospray ionization (ESI) source (capillary voltage: 3500 V), operating in the positive mode.
Samples (injection volume: 10 µL) were run in a mixture of methanol and water + 0.1 % formic acid (HPLC grade, Acros Organics) using a specific gradient program for each compound. For NRG (tR: 21.8 minutes), a linear gradient program was used, going from 40 % to 100 % methanol over a period of 40 minutes. For NRG-DM (tR: 31.2 minutes), a similar gradient was employed, starting initially at 50 % methanol and reaching 100 % over the same time period.
All samples were run at a flow rate of 0.12 mL/minute. Data were acquired and processed using Agilent LC/MSD ChemStation software rev. B.04.03-SP2 [105] . Further analysis was performed using Microsoft Excel 2016 and GraphPad Prism 5.
Samples (injection volume: 10 µL) were run in a mixture of methanol and water + 0.1 % formic acid (HPLC grade, Acros Organics) using a specific gradient program for each compound. For NRG (tR: 21.8 minutes), a linear gradient program was used, going from 40 % to 100 % methanol over a period of 40 minutes. For NRG-DM (tR: 31.2 minutes), a similar gradient was employed, starting initially at 50 % methanol and reaching 100 % over the same time period.
All samples were run at a flow rate of 0.12 mL/minute. Data were acquired and processed using Agilent LC/MSD ChemStation software rev. B.04.03-SP2 [105] . Further analysis was performed using Microsoft Excel 2016 and GraphPad Prism 5.
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