6545 q tof lc ms

Reactions were performed at room temperature for 30 min in 100 μL of 50 mM Tris pH 7.5, 1 mM 15, 4 mM sodium nitrite (or ¹⁵N-sodium nitrite), 5 mM ATP, 4 mM MgCl₂, and 100 μM Tri17. Chemically synthesized 15 was dissolved in DMSO to ensure full solubility and assays were maintained at a final concentration of 2% DMSO (vol/vol). After the incubation period, the reaction was quenched with two volumes of chilled methanol. The precipitated protein was removed by centrifugation (15,000 x g, 5 min) and the supernatant was used for analysis. LC-HRMS analysis was performed using an Agilent Technologies 6545 Q-TOF LC-MS equipped with an Agilent Eclipse Plus C18 column (4.6 × 100 mm). A water/acetonitrile mobile phase with 0.1% (vol/vol) formic acid with a linear gradient of 2-98% acetonitrile at a flow rate of 0.5 mL/min was utilized. 1 isolated from WT S. aureofaciens was utilized as a standard to compare the retention time, mass spectrum, and UV profiles between the biochemical assays. At least three independent replicates were performed for each assay, and representative results are shown. Analogous assays were performed with nitrate instead of nitrite and 2-HYAA or 12-aminododecanoic acid instead of 15, respectively.

Assays were performed in triplicate in 50 µL of 50 mM Tris pH 7.5 containing 5 mM nitrite, 5 mM ATP, 4 mM MgCl₂, and 20 µM Tri17. The tested concentrations for 15 were: 50 µM, 250 µM, 500 µM, 1 mM, and 2 mM. The incubation times for the reactions were 1 min, 5 min, 10 min, 20 min, and 40 min, which were used to determine the initial velocity of the reaction. After each incubation period, the reactions were quenched with two volumes of chilled methanol. The precipitated protein was removed by centrifugation (15,000 x g, 5 min) and the supernatant was used for analysis. LC-HRMS analysis was performed using an Agilent Technologies 6545 Q-TOF LC-MS equipped with an Agilent Eclipse Plus C18 column (4.6 × 100 mm). A water/acetonitrile mobile phase with 0.1% (vol/vol) formic acid with a linear gradient of 2-98% acetonitrile at a flow rate of 0.5 mL/min was utilized. Product concentration was estimated by comparing to authentic standard 1. Kinetic parameters were determined and plotted using GraphPad Prism.

Reactions were performed at room temperature for 30 min in 100 μL of 50 mM Tris pH 7.5, 1 mM lysine, 1 mM glycine, 2 mM NADPH, 0.2 mM FAD, 5 mM ATP, 2 mM MgCl₂, 20 μM Tri26, 20 μM Tri27, and 20 μM Tri28 (same concentration was used for dissected domains). The DNFB derivatization method was adapted from a previous work²⁵ . The reaction was then derivatized with 200 μL of 1 mM of DNFB dissolved in 200 mM borate pH 9.0 and incubated for 30 min at 60 °C. The precipitated protein was removed by centrifugation (15,000 x g, 5 min) and the supernatant was used for analysis. LC-HRMS analysis was performed using an Agilent Technologies 6545 Q-TOF LC-MS equipped with an Agilent Eclipse Plus C18 column (4.6 × 100 mm). A water/acetonitrile mobile phase with 0.1% (vol/vol) formic acid with a linear gradient of 2-98% acetonitrile at a flow rate of 0.5 mL/min was utilized. The same derivatization method was utilized with an authentic HAA standard. At least two independent replicates were performed for each assay, and representative results are shown.

Reactions were performed at room temperature for 1 hour in 100 μL of 50 mM HEPES pH 8.0, 2.5 mM CoA substrate, 2 mM MgCl₂, 100 μM ACP, 50 μM Tri14 and 50 μM Sfp. After the 1-hour incubation period, the reaction was quenched with two volumes of chilled methanol. The precipitated protein was removed by centrifugation (15,000 x g, 5 min) and the supernatant was used for analysis. LC-HRMS analysis was performed using an Agilent Technologies 6545 Q-TOF LC-MS equipped with an Agilent Eclipse Plus C18 column (4.6 × 100 mm). A water/acetonitrile mobile phase with 0.1% (vol/vol) formic acid with a linear gradient of 2-98% acetonitrile at a flow rate of 0.5 mL/min was utilized. Hexanoic and lauric acid standards were prepared and analyzed to test for hydrolytic activity of Tri14. Assays with hexanoyl-CoA and lauroyl-CoA were utilized with Tri20, while lauroyl-CoA was utilized solely for Tri30. At least three independent replicates were performed for each assay, and representative results are shown.

Tri17 assays utilizing nitrate, 2-HYAA, or 12-aminododecanoic acid were analyzed via LC-HRMS using an Agilent Technologies 6545 Q-TOF LC-MS equipped with an Agilent Eclipse Plus C18 column (4.6 × 100 mm). A water/acetonitrile mobile phase with 0.1% (vol/vol) formic acid with a linear gradient of 2-98% acetonitrile at a flow rate of 0.5 mL/min was utilized. Peak picking and comparative metabolomics were performed using MSDial (version 4.38) with peak lists exported to Microsoft Excel (Microsoft Office 365 Student).

Streptomycete cultures were pelleted by centrifugation (4,000 x g for 15 min), and the spent media were extracted 1:1 by volume with ethyl acetate. Ethyl acetate was removed by rotary evaporation, and the dry extract was dissolved in 300 μL of methanol for liquid chromatography-mass spectrometry (LC-MS) analysis by 10 μL injection onto an Agilent Technologies 6120 Quadrupole LC-MS instrument with an Agilent Eclipse Plus C18 column (4.6 × 100 mm). For high resolution MS (HRMS) analysis, the extract in methanol could be diluted by a factor of 5 for LC-HRMS and HRMS/MS analysis by 10 μL injection onto an Agilent Technologies 6545 Q-TOF LC-MS instrument with the same column. Linear gradients of 5-95% acetonitrile (vol/vol) in water with 0.1% formic acid (vol/vol) at 0.5 mL/min were used.