All peptides were purified using reverse-phase high pressure liquid chromatography on a Phenomenex Gemini C18 column (5 μm particle size) using a 2%–100% acetonitrile gradient program. To each solution was added 0.1% saturated NH4OH solution in water (for glutamic acid-containing sequences) or 0.1% trifluoroacetic acid (for lysine-containing sequences). System eluent was analyzed with an Agilent 6510 Q-TOF MS to identify product peaks. Acetonitrile was removed with rotary evaporation and water was removed via lyophilization. The resultant white, fluffy powder was stored at −20 °C.
6510 q tof ms
Manufactured by Agilent Technologies
Sourced in United States
The 6510 Q-TOF MS is a high-resolution mass spectrometer designed for accurate mass measurements and compound identification. It utilizes a Quadrupole-Time of Flight (Q-TOF) configuration to provide precise mass determination and high sensitivity.
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5 protocols using 6510 q tof ms
1
Peptide Purification and Characterization
2
Synthesis and Characterization of Novel Compounds
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Chemical reagents were purchased from Merck (Darmstadt, Germany) and used without further purification. Compounds 3 [22 (link)], 7 [23 (link)], 8 [22 (link)], 10 [24 (link)], 12 [24 (link)] and 13 [25 (link)] were prepared by procedures from the literature. Thin-layer chromatography was performed using Merck silica gel 60 F254 aluminium-backed plates. Reaction products were purified either using recrystallization or dry column vacuum chromatography on silica gel using gradient elutions as specified. NMR spectra were recorded on an Agilent 500 MHz or a Bruker 400 MHz spectrometer operating at 500 or 400 MHz for 1H NMR, respectively, and 125 or 100 MHz for 13C NMR, respectively. Spectra were referenced to the residual non-deuterated solvent signal using either CDCl3 (1H δ 7.26, 13C 77.00) or DMSO-d6 (1H δ 2.50, 13C δ 39.52). Multiplicity was assigned as singlet (s), doublet (d), triplet (t), quartet (q), quintet (quin), sextet (sxt) or multiplet (m). High-resolution mass spectra (HRMS) were recorded on an Agilent technologies 6510 Q-TOF MS. Compound purity was confirmed to be >95% prior to biological assays using quantitative NMR spectroscopy (see supplementary information ) [28 (link),29 (link)].
3
Analytical LC-QTOF-MS Characterization
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This study employed an Agilent Technologies (Santa Clara, CA, USA) 1290 Infinity II LC system coupled to an Agilent Technologies 6510 QTOF-MS using our previously developed methodology 8, (link)18 (link) . The chromatographic separation was carried out using a
4
Metabolite Identification by Mass Spectrometry
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For structural identification, we used the identical chromatographic conditions that were employed in the profiling experiment for the metabolite standards. Mass spectrometry using an Agilent 6510 Q-TOF MS was performed under the following conditions: gas temperature, 350 °C; flow rate of drying gas, 10 l/min; nebulizer pressure, 30 psig; capillary voltage, 4000 V; fragmentor voltage, 175 V; skimmer voltage, 65 V. MS and MS/MS spectra were both collected at 1.0 spectrum per second, with a medium isolation window of ~4 m/z. For MS/MS, the collision energy was set from 5 to 35 V. Several metabolites were further confirmed by an ion mobility mass spectrometer (SYNAPT HDMS, Waters) under similar chromatographic conditions.
5
Structural Identification of Metabolites
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We employed the same chromatographic conditions for structural identification as we did for the metabolite standards profiling experiment. The following parameters were used for mass spectrometry with an Agilent 6510 Q-TOFMS: gas temperature, 350 °C; flow rate of drying gas, 10 L/min; nebuliser pressure, 30 psig; capillary voltage, 4000 V; fragmentor voltage, 175 V; and skimmer voltage, 65 V. MS and MS/MS spectra were both collected at a rate of 1.0 spectrum per second with a medium isolation window of 4 m/z. The collision energy for MS/MS was varied from 5 to 35 V. An ion mobility mass spectrometer (SYNAPT HDMS, Waters) was used to confirm several metabolites under identical chromatographic conditions.
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