The chemicals and reagents employed were of LR grade and procured from Sigma Aldrich (Mumbai, India), E. Merck (Mumbai, India), S.D. Fine Chemicals Ltd. (Mumbai, India), and Qualigens (New Delhi, India). Thin-layer chromatography (TLC) was carried out to observe the advancement of the reactions using benzene and acetone (80:20, v/v), as well as toluene/ethyl acetate/formic acid (50:40:10, v/v/v) as a solvent system, and spots were traced using iodine vapor or UV-light (254 nm). Melting points of the synthesized molecules were calculated using an electrical melting point apparatus (open capillary method) and are uncorrected. The infrared spectra were noted in the region from the 4000 to 400 cm−1 range on the Shimadzu FT-IR spectrometer, while 1H and 13C-NMR spectra were recorded on the Bruker Advance-500 MHz and 125 MHz spectrometer using DMSO-d6 or CDCl3 as an NMR solvent. Synapt-mass spectrometric detection was performed on UPLC-MS (Q-TOF-ESI; Waters Corp., Milford, MA, USA) using the ESI technique. Elemental analysis was executed on the CHNOS-Elemental analyzer (Vario EL III) and found to be ±0.4%, i.e., within the theoretical values.
Esi q tof
Manufactured by Waters Corporation
Sourced in United States
The ESI-Q-TOF is a high-performance liquid chromatography-mass spectrometry (LC-MS) system developed by Waters Corporation. It combines an electrospray ionization (ESI) source with a quadrupole-time-of-flight (Q-TOF) mass analyzer, providing accurate mass measurements and high-resolution data for the analysis of a wide range of analytes, including biomolecules and small molecules.
Automatically generated - may contain errors
Lab products found in correlation
Synapt g2 s hdms, by Waters Corporation (2 mentions)
Advance 500 mhz, by Bruker (1 mentions)
Ftir spectrometer, by Bruker (1 mentions)
300 mhz nmr instrument, by Bruker (1 mentions)
Ft ir spectrometer, by Shimadzu (1 mentions)
Synapt g2, by Waters Corporation (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Mascot, by Matrix Science (1 mentions)
Chymotrypsin, by Roche (1 mentions)
Ppsq 31a protein sequencer, by Shimadzu (1 mentions)
Synapt hdms, by Waters Corporation (1 mentions)
Trypsin, by Promega (1 mentions)
9 protocols using esi q tof
1
Synthesis and Characterization of Organic Compounds
2
UPLC-MS/MS Analysis of Plant Metabolites
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JA, SA and camalexin were extracted and their levels determined by UPLC-MS/MS (Q-ToF-ESI; Synapt G2-S HDMS; Waters®, Milford, Massachusetts, United States) as described in Valsamakis et al. (2020) (link).
3
Quantifying SA and Camalexin in Plant Leaves
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Leaf material from A. thaliana, S. dulcamara and U. minor was analyzed for SA concentrations. Additionally, camalexin concentrations were determined in leaf material from A. thaliana. SA and camalexin were extracted similar as described by Wang et al.72 (link). We used untreated leaves, leaves locally treated for three days with egg extracts or leaves with natural egg deposition (for details on the extraction procedure, compare Supplementary material: Methods).
We used UPLC-MS/MS (Q-ToF-ESI; Synapt G2-S HDMS; Waters®, Milford, Massachusetts) for separation, detection and quantification of SA and camalexin. We applied the methods as described by Valsamakis et al.40 (link) (further details in Supplementary material: Methods).
We used UPLC-MS/MS (Q-ToF-ESI; Synapt G2-S HDMS; Waters®, Milford, Massachusetts) for separation, detection and quantification of SA and camalexin. We applied the methods as described by Valsamakis et al.40 (link) (further details in Supplementary material: Methods).
4
Synthesis and Characterization of Organic Compounds
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Chemicals
and reagents
were purchased from Sigma-Aldrich (India), Merck, and S. D. Fine Chemicals.
Thin-layer chromatography (TLC) was used for the assessments of the
confirmation of the reaction and purity of the compounds. Melting
points (M.P.) of the synthesized compounds were obtained by using
open capillary tubes on a melting point apparatus. The infrared (IR)
spectrum was recorded using a Shimadzu FT-IR spectrometer in the 4000–400
cm–1 range by using KBr (potassium bromide) pellets.
The Bruker 300 MHz NMR instrument was used for the recording of the 1H-NMR spectrum of compounds in the solvent DMSO-d6. UPLC–MS (Q-TOF-ESI) (Waters Corp., USA) with
an ESI method was performed for molecular mass detection (m/z) and purity of the synthesized compounds.
and reagents
were purchased from Sigma-Aldrich (India), Merck, and S. D. Fine Chemicals.
Thin-layer chromatography (TLC) was used for the assessments of the
confirmation of the reaction and purity of the compounds. Melting
points (M.P.) of the synthesized compounds were obtained by using
open capillary tubes on a melting point apparatus. The infrared (IR)
spectrum was recorded using a Shimadzu FT-IR spectrometer in the 4000–400
cm–1 range by using KBr (potassium bromide) pellets.
The Bruker 300 MHz NMR instrument was used for the recording of the 1H-NMR spectrum of compounds in the solvent DMSO-d6. UPLC–MS (Q-TOF-ESI) (Waters Corp., USA) with
an ESI method was performed for molecular mass detection (m/z) and purity of the synthesized compounds.
5
IM-MS Analysis of Lasso Peptides
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IM-MS experiments were carried out on a hybrid quadrupole ESI-Q-TOF IM-MS instrument (Synapt G2, Waters). The pre-purified peptides 9401-LP1 and Snou-LP, as well as the lasso and non-lasso peptide controls, MccJ25 and MccJ25-lcm, were solubilized in H2O/CH3CN 1:1 + 0.1% formic acid +0.5% sulfolane. The solutions were introduced in the source by direct infusion using a syringe pump with a flow rate of 300 μL/h. Ionization was operated in positive ion mode under the experimental set-up as described39 (link). The CCS calibration was carried out from the CCS values of doubly-protonated polyalanine ions determined with uniform field drift tube ion mobility with helium as drift gas55 (link). The 9810-LP was in too low quantity/purity to provide information on the peptide topology by IM-MS.
6
Tandem Mass Spectrometry Protein ID
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To confirm that the DNA sequence identified by the 3ʹ RACE and genome walking encodes the short-style–specific protein, we obtained internal partial amino acid sequences de novo by tandem mass spectrometry (MS/MS) with ESI-Q-TOF (Waters, Milford, USA).
7
Protein Separation and Identification
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Samples were separated on a NuPAGE 4%–12% Bis-Tris gel (Thermo Fisher Scientific, Carlsbad, CA, USA) and colloidal Coomassie stained. Lanes were divided in 23 fragments that were in gel digested with trypsin, extracted (42 (link)) and peptides were analyzed in a capillary HPLC coupled electrospray ionization quadrupole time of flight (ESI-Q-TOF, Ultima, Waters, Milford, MA, USA) mass spectrometer as described (28 (link)). Peptide assignments and data analysis were performed by Mascot, Matrix Science (London, UK) (28 (link)).
8
Detecting Protein-Ligand Molecular Weights
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The molecular weights of protein molecules and the protein–ligand adducts were detected as suggested by others (Böth et al., 2013 (link)). In brief, 5 mg of Cdc25B (372–551) was incubated with or without 1 mM HB-21 at 20°C for 30 min. Subsequently, the samples were diluted in 0.5 ml denaturing buffer [5%(v/v) acetonitrile, 0.1% (v/v) formic acid, 0.5 mM TCEP], and the molecular weights were detected by ESI-Q-TOF (Waters Corp.).
9
Protein Sequencing by Edman Degradation and MS/MS
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Automated Edman degradations were performed in the Shimadzu model PPSQ-31A protein sequencer (Shimadzu Corp., Japan). PTH-amino acids from the N-terminus sequence were separated on a 2.0 × 250 mm Wakosil ODS column (Wako Pure Chemical Corp., Osaka, Japan) connected to a model LC-20AT pump. The absorbance was detected at 269 nm with a UV-Vis SPD-20A detector.
A MS/MS approach was employed to determine internal sequences. First, an SDS-PAGE was conducted, as described above. Then, protein spots were sliced, discolored, reduced and carboxyamidomethylated, as described by Shevchenko et al. (2006) . After that, protein spots were digested with trypsin (Promega, WI, USA) and chymotrypsin (Roche, Switzerland) at ratio of 1:50 (enzyme:substrate) for 16 h at 37°C. Peptides were extracted and concentrated, as described by Shevchenko et al. (2006) . Cienc (2023) 95(1) e20220379 4 | 14 The peptides were separated on a reverse phase C18 nanocolumn (0.075 x 100 mm) coupled to a nanoAcquity system. The eluates were directly infused in a hybrid mass spectrometer (ESI-Q-ToF) (Synapt HDMS, Waters Corp, MA, USA). The instrument parameters were adjusted, as described by Carneiro et al. (2013a) . MS/ MS spectra were interpreted manually, and sequenced peptides were searched online against NCBI and UniProt databanks.
A MS/MS approach was employed to determine internal sequences. First, an SDS-PAGE was conducted, as described above. Then, protein spots were sliced, discolored, reduced and carboxyamidomethylated, as described by Shevchenko et al. (2006) . After that, protein spots were digested with trypsin (Promega, WI, USA) and chymotrypsin (Roche, Switzerland) at ratio of 1:50 (enzyme:substrate) for 16 h at 37°C. Peptides were extracted and concentrated, as described by Shevchenko et al. (2006) . Cienc (2023) 95(1) e20220379 4 | 14 The peptides were separated on a reverse phase C18 nanocolumn (0.075 x 100 mm) coupled to a nanoAcquity system. The eluates were directly infused in a hybrid mass spectrometer (ESI-Q-ToF) (Synapt HDMS, Waters Corp, MA, USA). The instrument parameters were adjusted, as described by Carneiro et al. (2013a) . MS/ MS spectra were interpreted manually, and sequenced peptides were searched online against NCBI and UniProt databanks.
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