The freeze-dried HPLC elutes possessing antifungal activity were dissolved in methanol for LC-MS/MS analysis. The C18 column (Waters UPLC ACQUITY UPLC BEH C18 1.8 μm, 100 mm × 2.1 mm, Waters, Milford, MA, USA) was used with the following gradients: 0–6 min at 5–99.5% of ACN + 0.1% formic acid, 6–8 min at 99.5% of ACN + 0.1% formic acid, 8–8.2 min at 99.5%–5% of ACN + 0.1% formic acid and 8.2–10 min at 5% of ACN + 0.1% formic acid. The rate of flow was 0.4 mL/min. The mass data were analyzed by the Thermo Orbitrap Elite system, and the profile mode was set to positive mode with a mass range of m/z 50–1500. The resolution of Orbitrap was 60,000 resolution (MS1), 15,000 (DDA), and 30,000 (DDA2). For tandem mass (MS/MS), the top five intense ions from each full mass scan were selected for collision-induced dissociation fragmentation (CID). Then the fragments from MS/MS data were used to predict the structures.
Orbitrap elite system
Manufactured by Thermo Fisher Scientific
The Orbitrap Elite system is a high-resolution mass spectrometer designed for advanced analytical applications. It utilizes the Orbitrap mass analyzer, which provides accurate mass measurements and high-resolution capabilities. The core function of the Orbitrap Elite system is to perform sensitive and precise mass analysis of a wide range of molecular analytes.
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Lab products found in correlation
Ziptip, by Merck Group (1 mentions)
Bca assay, by Beyotime (1 mentions)
Total protein extraction kit, by BestBio (1 mentions)
Xcalibur qual browser, by Thermo Fisher Scientific (1 mentions)
Prism 8, by GraphPad (1 mentions)
Human serum, by Lonza (1 mentions)
Vortex genie 2, by Scientific Industries (1 mentions)
Speedvac concentrator, by Thermo Fisher Scientific (1 mentions)
5 protocols using orbitrap elite system
1
Profiling Antifungal Metabolites by LC-MS/MS
2
Quantitative Proteomic Analysis of K150 Cells
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K150 cells (1 × 107 per sample) were treated using a Total Protein Extraction Kit (BestBio, Shanghai, China), and protein concentration was measured using a BCA assay (Beyotime). Protein (100 µg in 10 µl) was incubated with 50 µl of denaturation buffer [0.5 M Tris-HCl (pH 8.1 ± 0.1), 2.75 mM EDTA, 6 M guanidine-HCl]. Then 30 µl of 1 M DTT was added, the mixture was incubated at 37 °C for 2 h, 50 µl of 1 M iodoacetamide was added, and the mixture was incubated in the dark at room temperature for 1 h. Samples were concentrated by centrifugation on YM-3 ultrafiltration filters (Millipore, MA, USA), then washed four times with HPLC water. Samples were incubated at 37 °C overnight with 4 µg of trypsin, and purified through C18 pipette tip filters (ZipTip, Millipore). Proteins were identified and quantified by liquid chromatography and mass spectrometry on an Orbitrap Elite system (ThermoFisher) at Shandong University.
3
Angie1 Stability in Human Serum
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Human serum (14-490E, Lonza, Basel, Switzerland) was spiked with 10 μM Angie1 and incubated at 37°C for 2 h in triplicates. After 5, 10, 20, 30, 45, 60, 90, and 120 min; aliquots (5 μl) were diluted with 1.5 ml ice cold TFA (0.1%). Around 15 μl of each sample was analyzed using an Orbitrap Elite system (Thermo Fisher Scientific) as described above. XCalibur Qual Browser (Thermo Fisher Scientific) was used for visualization of the total ion chromatograms and spectra, as well as for signal area calculation. The theoretical spectrum of Angie1 and its degradation products were generated and the highest intensity signals were used as reference for detecting Angie1 and its degradation products. Plot graphs (signal area vs. time) and half-lives were calculated with GraphPad Prism 8.0 (GraphPad, La Jolla, CA, United States).
4
Quantification of L-4-nitro-Trp by LC-MS
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After cell-free biosynthesis, an equal volume of 100% methanol (i.e., 180 μL methanol per reaction) was added to the reaction mixture to terminate the cell-free reaction. The resulting solution was mixed thoroughly for 1 min by vortex (VORTEX-GENIE2, Scientific Industries). Afterward, the mixture was centrifuged for 15 min at 21,000 g and the resulting supernatant was directly used for product analysis by LC-MS. All experiments were repeated three times.
L-4-nitro-Trp quantification was performed using an Agilent 1260 LC-MS System equipped with an Agilent Proshell 120 EC-C18 column (3.0 × 50 mm, 2.7 μm) coupled with a ThermoFisher Orbitrap Elite system. For L-4-nitro-Trp detection, 2 μL of each sample was injected and separated at a flow rate of 0.3 mL/min and 40 °C column temperature. Water (A) and acetonitrile (B) each containing 0.1% formic acid were used as mobile phases with a linear gradient program (10–90% solvent B) for 15 min to separate chemicals. A pre-wash phase of 10 min with 10% solvent B was added at the beginning of each run. For MS/MS analysis, MS was acquired under a Full-Scan mode in a mass range of m/z 150–1000. Fragmentation was introduced by high energy collision-induced dissociation (HCD) technique with normalized collision energy at 35 eV. MS/MS resolution was 15,000 and the scan range was 50–300.
L-4-nitro-Trp quantification was performed using an Agilent 1260 LC-MS System equipped with an Agilent Proshell 120 EC-C18 column (3.0 × 50 mm, 2.7 μm) coupled with a ThermoFisher Orbitrap Elite system. For L-4-nitro-Trp detection, 2 μL of each sample was injected and separated at a flow rate of 0.3 mL/min and 40 °C column temperature. Water (A) and acetonitrile (B) each containing 0.1% formic acid were used as mobile phases with a linear gradient program (10–90% solvent B) for 15 min to separate chemicals. A pre-wash phase of 10 min with 10% solvent B was added at the beginning of each run. For MS/MS analysis, MS was acquired under a Full-Scan mode in a mass range of m/z 150–1000. Fragmentation was introduced by high energy collision-induced dissociation (HCD) technique with normalized collision energy at 35 eV. MS/MS resolution was 15,000 and the scan range was 50–300.
5
Lipid Extraction and LC-MS/MS Analysis
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Cell pellets were washed twice with PBS and resuspended in methanol. After spiking with 30 ml of lipid standards mixture of 400 ng 17:0-CE, 1,000 ng CH-d7, 100 ng 17:0-FACoA, 400 ng 17:0-FFA, and 50 ng C 17 -Cer, LC-MSgrade water and chloroform were added. The mixture was subjected to Folch extraction. After collection of the lower phase, the upper phase was re-extracted with synthetic lower phase. The lower phase from both extractions was combined and dried at room temperature with a Speed-Vac concentrator (Thermo Scientific). The lipids were re-dissolved in chloroform and injected for LC-MS/MS analysis (Orbitrap Elite system, Thermo Fisher).
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