6540 uhd accurate mass q tof

Manufactured by Agilent Technologies

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The 6540 UHD Accurate-Mass Q-TOF is a high-resolution quadrupole time-of-flight mass spectrometer designed for precise mass measurements and identification of compounds. It utilizes a quadrupole analyzer and a time-of-flight mass analyzer to provide accurate mass data for unknown compounds.

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10 protocols using 6540 uhd accurate mass q tof

UPLC-QTOF-MSMS Protocol for Metabolomics

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Full scan /automated MSMS in data dependent mode was acquired in both negative and positive modes for all files (100- 1400 m/z) using a UPLC system coupled online with a 6540 UHD Accurate-Mass Q-TOF (Agilent Technologies) following exact conditions described in Saied & Farag (2023) (link) and Sallam et al. (2022) (link). Exact details are described in the Supplemental File. Molecular networks were generated for negative and positive ionization files applying Global Natural Products Social Molecular networking (GNPS, http://gnps.ucsd.edu/ProteoSAFe/status/gnps-splash.jsp) accessed date (14 April 2022). The parameters are mentioned in the Supplemental File.

Mostafa M.M, & Farag M.A. (2023). Profiling of primary and phytonutrients in edible mahlab cherry (Prunus mahaleb L.) seeds in the context of its different cultivars and roasting as analyzed using molecular networking and chemometric tools. PeerJ, 11, e15908.

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Metabolite Fingerprinting by UHPLC-HRMS

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Metabolite fingerprinting was conducted according to Feussner et al. (2022) (link) as described in Mohnike et al. (2021) (link). In brief, extracted samples were analyzed with the UHPLC1290 (Agilent Technologies, Santa Clara, CA, USA) coupled to an HRMS instrument 6540 UHD Accurate Mass Q-TOF (Agilent Technologies) with Agilent Dual Jet Stream Technology as the ESI source (Agilent Technologies). An ACQUITY HSS T3 column (2.1 × 100 mm, 1.8 µm particle size, Waters Corporation) was used for chromatographic separation at a flow rate of 500 µl min^–1 at 40 °C. The solvent system applied was A [water, 0.1% (v/v) formic acid] and B [acetronitrile, 0.1% (v/v) formic acid]. The gradient applied was: 0–3 min, 1–20% B; 3–8 min, 20–97% B; 8–12 min, 10% B; 12–15 min, 1% B. The technical details were described recently (Mohnike et al., 2021 (link)). Data were acquired using Mass Hunter Acquisition B.03.01. Data deconvolution was performed using Profinder 10.0 (Agilent Technologies). Data were processed using MarVis-Suite (Kaever et al., 2009 (link), 2012 (link), 2015 (link)) (http://marvis.gobics.de) or OriginPro2020 (OriginLab Corporation, Northampton, MA, USA).

Mohnike L., Huang W., Worbs B., Feussner K., Zhang Y, & Feussner I. (2022). N-Hydroxy pipecolic acid methyl ester is involved in Arabidopsis immunity. Journal of Experimental Botany, 74(1), 458-471.

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Targeted Metabolomics by LC-QTOF

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The liquid chromatography–mass spectrometry was performed on a 1290 Infinity Binary UPLC coupled with a 6540 UHD Accurate-Mass Q-TOF (Agilent Technologies, Santa Clara, CA, USA) as described previously by Hanhineva et al.^{43 (link)} Briefly, a Zorbax Eclipse XDB-C18 column was used for the reversed-phase separation and an Aqcuity UPLC BEH amide column (Waters, Milford, MA, USA) for the HILIC separation. After each chromatographic separation, the ionization was carried out using jet stream electrospray ionization (ESI) in the positive and negative mode, yielding four data files per sample. The collision energies for the MS/MS analysis were chosen as 10, 20 and 40 V, for compatibility with the spectral databases.

Koistinen V.M., Mattila O., Katina K., Poutanen K., Aura A.M, & Hanhineva K. (2018). Metabolic profiling of sourdough fermented wheat and rye bread. Scientific Reports, 8, 5684.

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Isotopic Labeling of Lactate

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First, 200 nmol of HadD AJ1 in 25 mM Tris-HCl, pH 8.0, were lyophilized. The reaction was initiated by dissolving the dried enzyme in 300 μL H₂¹⁸O containing 39 mM Glycine-NaOH, pH 10.0, and 20 nmol D-2-CPA. The reaction mixture was ultrafiltered after the incubation at 30 °C for 18 h and was then directly injected into the mass spectrometer. The molecular mass of the produced lactate was measured in the quadrupole time-of-flight mass spectrometry (Agilent Technologies 6540 UHD Accurate-Mass Q-TOF, USA) equipped with an electrospray ionization ion source in the negative mode.

Wang Y., Feng Y., Cao X., Liu Y, & Xue S. (2018). Insights into the molecular mechanism of dehalogenation catalyzed by D-2-haloacid dehalogenase from crystal structures. Scientific Reports, 8, 1454.

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Comprehensive LC-MS Metabolite Profiling

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The samples were analyzed by liquid chromatography-mass spectrometry consisting of a 1290 Infinity Binary UPLC coupled with a 6540 UHD Accurate-Mass Q-TOF (Agilent Technologies), as described previously [49 (link)]. In brief, a Zorbax Eclipse XDB-C18 column (2.1 × 100 mm, 1.8 μm; Agilent Technologies) was used for the reversed-phase (RP) separation and an Acquity UPLC BEH amide column (Waters) for the HILIC separation. After each chromatographic run, the ionization was carried out using jet stream electrospray ionization (ESI) in the positive and negative mode, yielding four data files per sample. The collision energies for the MS/MS analysis were selected as 10, 20, and 40 V, for compatibility with spectral databases.

Ni Y., Lohinai Z., Heshiki Y., Dome B., Moldvay J., Dulka E., Galffy G., Berta J., Weiss G.J., Sommer M.O, & Panagiotou G. (2021). Distinct composition and metabolic functions of human gut microbiota are associated with cachexia in lung cancer patients. The ISME Journal, 15(11), 3207-3220.

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Metabolite Profiling of Crude Extracts

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High-resolution electrospray ionization mass spectra (HRESIMS) were obtained on an Agilent 6230A LC/TOF, and tandem mass spectra were obtained on an Agilent 6540 UHD Accurate-Mass QTOF LC/MS, each in positive ionization mode, with samples dissolved in MeOH and eluted with a gradient of H₂O to ACN on a Phenomenex Kinetex C-18 column (2.6 μm, 100 Å, 150 × 3 mm). LC-MS/MS data was processed using Agilent MassHunter Qualitative Analysis B.05.00. Compounds were identified and spectra collected into a profile with software automated Compound Finder (Auto MS/MS) and MS/MS Spectral Extraction. All compounds within an extract were analyzed with public databases, METLIN-AMRT-PCDL and Mycotoxins-AMRT-PCDL, and our own in-house database, using MassHunter Qualitative Analysis as specified by manufacturer's instructions.
HRESIMS and liquid chromatography/tandem MS (LC-MS/MS) spectral profiles were generated for each crude extract. Extracts were subjected to LC-MS with a standardized elution gradient to generate a metabolite chromatographic and HRESIMS profile. Crude extracts were then subjected to an automated LC-MS/MS experiment which utilized the same standardized elution gradient as the LC-MS experiment, with collision-induced dissociation (CID) energies of 0 mV, 10 mV, and 40 mV selected to match data provided in most public databases [2 (link), 9 (link), 14 ].

Knestrick M.A., Tawfik R., Shaw L.N, & Baker B.J. (2019). Chromatographic Editing Enhances Natural Product Discovery. Journal of pharmaceutical and biomedical analysis, 176, 112831.

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LC-MS Metabolomics Protocol for Diverse Samples

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The samples were analyzed by liquid chromatography–mass spectrometry (LC-MS), consisting of a 1290 Infinity Binary UPLC coupled with a 6540 UHD Accurate-Mass Q-TOF (Agilent Technologies Inc., Santa Clara, CA, USA), as described previously [63 (link)]. In brief, a Zorbax Eclipse XDB-C18 column (2.1 × 100 mm, 1.8 μm; Agilent Technologies) was used for the reversed-phase (RP) separation and an Acquity UPLC BEH amide column (Waters Corporation, Milford, MA, USA) for the HILIC separation. After each chromatographic run, the ionization was carried out using jet stream electrospray ionization (ESI) in the positive and negative mode, yielding four data files per sample. The collision energies for the MS/MS (tandem mass spectrometry) analysis were selected as 10, 20 and 40 V, for compatibility with spectral databases.

Pessa-Morikawa T., Husso A., Kärkkäinen O., Koistinen V., Hanhineva K., Iivanainen A, & Niku M. (2022). Maternal microbiota-derived metabolic profile in fetal murine intestine, brain and placenta. BMC Microbiology, 22, 46.

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Kinetic Analysis of UGT76B1 Substrates

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UGT76B1 recombinant protein was incubated with substrates NHP, SA, and ILA for 30 min at 30°C. The reaction was stopped by the addition of 20% acetonitrile. Samples were analyzed using a 1290 Infinity UHPLC system coupled to a 6540 UHD Accurate-Mass Q-TOF (Agilent Technologies, Santa Clara, CA, USA) as previously described (Feussner and Feussner, 2019 ). Kinetic parameters of UGT76B1’s substrates NHP, SA, and ILA were analyzed as described under UPLC-nanoESI-QTRAP-MS-based metabolite quantification. The reaction mixture contained 3.5-µg UGT76B1, 2 mM UDP-Glc (Merck, Darmstadt, Germany), and 0–2.5 mM substrate. Before incubation with UGT76B1, the initial amount of substrate was determined for analysis of substrate reduction. The reaction was incubated for 15 min at 30°C and stopped by the addition of methanol. The difference in signal intensity of substrate was plotted for each substrate and concentration. The K_M was determined via Hill regression analysis using OriginPro version 8.5 (OriginLab Corporation, Northampton, MA, USA).

Mohnike L., Rekhter D., Huang W., Feussner K., Tian H., Herrfurth C., Zhang Y, & Feussner I. (2021). The glycosyltransferase UGT76B1 modulates N-hydroxy-pipecolic acid homeostasis and plant immunity. The Plant Cell, 33(3), 735-749.

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Kinetic Analysis of UGT76B1 Substrates

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UGT76B1 recombinant protein was incubated with substrates NHP, SA and ILA for 30 min at 30 °C. The reaction was stopped by the addition of 20 % acetonitrile. Samples were analyzed using a 1290 Infinity UHPLC system coupled to a 6540 UHD Accurate-Mass Q-TOF (Agilent Technologies, USA) as previously described (Feussner and Feussner, 2019) . Kinetic parameters of UGT76B1's substrates NHP, SA and ILA were analyzed via UHPLC-HRMS. The reaction mixture contained 3.5 µg UGT76B1, 2 mM UDP-Glc (Merck KGaA) and 0-2.5 mM substrate. Before the incubation with UGT76B1, the initial amount of substrate was determined for analysis of substrate reduction. The reaction was incubated for 15 min at 30 °C and stopped by the addition of MeOH. The difference in signal intensity of substrate was plotted for each substrate and concentration. The Michaelis-Menten constant KM was determined via Hill regression analysis using OriginPro8.5 (OriginLab Corporation, Northampton, MA, USA).

Mohnike L., Rekhter D., Huang W., Feussner K., Tian H., Herrfurth C., Zhang Y., & Feußner I. (2020). The glycosyltransferase UGT76B1 is critical for plant immunity as it governs the homeostasis of N-hydroxy-pipecolic acid.

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Quantitative Metabolomic Analysis by LC-MS

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The samples were analyzed by liquid chromatography-mass spectrometry, consisting of a 1290 Infinity Binary UPLC coupled with a 6540 UHD Accurate-Mass Q-TOF (Agilent Technologies Inc., Santa Clara, CA, USA), as described previously (Klåvus et al., 2020) (link). In brief, a Zorbax Eclipse XDB-C18 column (2.1 × 100 mm, 1.8 µm; Agilent Technologies) was used for the reversed-phase (RP) separation and an Acquity UPLC BEH amide column (Waters Corporation, Milford, MA, USA) for the HILIC separation. After each chromatographic run, the ionization was carried out using jet stream electrospray ionization (ESI) in the positive and negative mode, yielding four data files per sample. The collision energies for the MS/MS analysis were selected as 10, 20 and 40 V, for compatibility with spectral databases.

Pessa‐Morikawa T., Husso A., Kärkkäinen O., Koistinen V., Hanhineva K., Iivanainen A., & Niku M. (2020). Metabolomic signature of the maternal microbiota in the fetus.

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