Multidomain peptides were designed based on previously published sequences from our laboratory: SL: K2(SL)6K2 and SLac: K(SL)2(SLRG)(SL)3K(GRGDS) [6 (link)]. All peptides, resin and coupling reagents were purchased from Aapptec (Louisville, KY). Standard solid phase peptide synthesis was performed on an Apex Focus XC (Aapptec), using Rink amide resin with 0.37mM loading and N-terminal acetylation. Post cleavage from resin, peptides were dialyzed with 500–1200 MWCO dialysis tubing (Sigma-Aldrich, St. Louis, MO) against DI water. Peptides were subsequently lyophilized, confirmed for purity using electron-spray ionization mass spectrometry, MicroTOF ESI (Bruker Instruments, Billerica, MA), and reconstituted at 20 mg/mL in sterile 298 mM sucrose. Gelation of peptide was achieved by addition of volume equivalents of pH 7.4 buffer with 1X PBS or HBSS. For certain studies comparison to biosynthetic scaffolds was performed. Scaffolds used include: acid solubilized Type I rat tail tendon collagen (4.0 mg/mL neutralized), Matrigel™ (8.2 mg/mL) and tissue culture polystyrene (TCP) which were all obtained from BD Biosciences (Franklin Lakes, NJ).
Microtof esi
Manufactured by Bruker
Sourced in Germany
The MicroTOF ESI is a compact time-of-flight mass spectrometer that uses electrospray ionization (ESI) for sample introduction. It is designed to provide accurate mass measurements and high-resolution mass spectra for a wide range of analytes, including small molecules, peptides, and proteins.
Automatically generated - may contain errors
Lab products found in correlation
Apex focus xc, by AAPPTec (2 mentions)
Opus ft ir spectrometer, by Bruker (2 mentions)
6110 hplc ms esi, by Agilent Technologies (2 mentions)
Mwco dialysis tubing, by Merck Group (1 mentions)
Matrigel, by BD (1 mentions)
Ultraviolet 1800 spectrophotometer, by Shimadzu (1 mentions)
Silica gel 60 f254, by Merck Group (1 mentions)
1260 infinity quaternary lc, by Agilent Technologies (1 mentions)
Avance 500 mhz, by Bruker (1 mentions)
Agilent 1100 series instrument, by Agilent Technologies (1 mentions)
Avance 2 400 mhz, by Bruker (1 mentions)
Aviii400, by Bruker (1 mentions)
Av 400, by Bruker (1 mentions)
Dpx 400, by Bruker (1 mentions)
Lc20 prominence hplc system, by Grace Bio-Labs (1 mentions)
C18 vydac, by Grace Bio-Labs (1 mentions)
Thiosemicarbazide, by Merck Group (1 mentions)
4 nitrobenzaldehyde, by Merck Group (1 mentions)
Luna c18 reversed phase column, by Phenomenex (1 mentions)
Compass dataanalysis 4, by Bruker (1 mentions)
10 protocols using microtof esi
1
Multidomain Peptide Hydrogel Fabrication
2
Synthesis and Characterization of Thiosemicarbazones
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All reagents used in synthesis and biological evaluation were commercial (Merck, Fischer and Acros Organics) and were used as supplied. Initial thiosemicarbazones (1) are readily available either commercially and via simple one-step literature procedures (see ref. 17–25 (link)). Chromatographic silica gel 60 (220–240 mesh) was obtained from Merck. Thin layer chromatography was performed on foil-backed plates coated with Merck Silica gel 60 F254. Plates were developed using ultraviolet light and basic aqueous potassium permanganate. Uncorrected melting points were measured on a DMP-300 A&E Lab apparatus. Ultraviolet (UV) absorption spectra were recorded on Shimadzu Ultraviolet-1800 spectrophotometer in the range 200–800 nm. Infrared spectra were recorded on a Bruker OPUS FT-IR spectrometer by attenuated total reflection (diamond-ATR) on solid films. Proton and 13C NMR spectra were recorded on Bruker DPX-400 and 500 MHz spectrometers at ambient temperatures; where necessary HSQC and HMBC techniques were used to confirm structural connectivities. Mass spectra were recorded on Bruker Micro TOF-ESI positive targeted mode. Elemental (CHN) analyses were conducted on a CE-440 Elemental Analyzer.
3
General Organic Chemistry Techniques
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All reagents were purchased from
commercial suppliers and used without further purification. Reactions
were carried out in 4 mL screw neck glass vials furnished with screw
caps equipped with poly(tetrafluoroethylene) (PTFE)/rubber septa,
and stir bars under ambient atmosphere unless otherwise noted. Silica
gel 60 Å (40–60 μm, 230–400 mesh) was used
for column chromatography. All NMR spectra were recorded in CDCl3 using a Bruker AVANCE II 400 MHz or Bruker Avance 500 MHz.
Chemical shifts are given in ppm relative to the residual solvent
peak (1H NMR: CDCl3 δ 7.26, 13C NMR: CDCl3 δ 77.16) with multiplicity (br = broad,
s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet),
coupling constants (in hertz), and integration. Kinetic data was analyzed
by Agilent 1260 Infinity Quaternary LC (Eclipse Plus 18C column, 3.5
μm, 4.6 × 100 mm2; UV detector, 265 nm) with
a gradient of acetonitrile and 0.1% formic acid in Milli-Q water at
a flow rate of 1 mL/min. The analytes were calibrated using a five-point
calibration curve with threefold dilution between each sample in the
series. HPLC with a chiral stationary phase was performed on an Agilent
1100 series instrument. High-resolution mass spectrometry analyses
were performed by Thermo Scientific Q Exactive HF Hybrid Quadrupole-Orbitrap
HESI or Bruker microTOF ESI, and low-resolution mass analyses by Bruker
Daltonics amaZon speed no 06052 ESI.
commercial suppliers and used without further purification. Reactions
were carried out in 4 mL screw neck glass vials furnished with screw
caps equipped with poly(tetrafluoroethylene) (PTFE)/rubber septa,
and stir bars under ambient atmosphere unless otherwise noted. Silica
gel 60 Å (40–60 μm, 230–400 mesh) was used
for column chromatography. All NMR spectra were recorded in CDCl3 using a Bruker AVANCE II 400 MHz or Bruker Avance 500 MHz.
Chemical shifts are given in ppm relative to the residual solvent
peak (1H NMR: CDCl3 δ 7.26, 13C NMR: CDCl3 δ 77.16) with multiplicity (br = broad,
s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet),
coupling constants (in hertz), and integration. Kinetic data was analyzed
by Agilent 1260 Infinity Quaternary LC (Eclipse Plus 18C column, 3.5
μm, 4.6 × 100 mm2; UV detector, 265 nm) with
a gradient of acetonitrile and 0.1% formic acid in Milli-Q water at
a flow rate of 1 mL/min. The analytes were calibrated using a five-point
calibration curve with threefold dilution between each sample in the
series. HPLC with a chiral stationary phase was performed on an Agilent
1100 series instrument. High-resolution mass spectrometry analyses
were performed by Thermo Scientific Q Exactive HF Hybrid Quadrupole-Orbitrap
HESI or Bruker microTOF ESI, and low-resolution mass analyses by Bruker
Daltonics amaZon speed no 06052 ESI.
4
Mass Spectrometry Analysis of Dye Extracts
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Mass spectroscopy of all the dye extracts was determined in methanol and water using MicroTof‐ESI (Bruker Daltonics) and Orbitrap LTQ XL‐ESI (Thermo Fisher Scientific) in order to assess the effect of the temperature conditions and solvents used for the extractions on the compositions of the extracts. Additionally, the fractionization and mass spectroscopy of the dye extract (CM) was done using 6110 HPLC‐MS‐ESI (Agilent Technologies). In all cases, both positive and negative ionization modes of the mass spectroscopy were acquired.
5
Characterization of Organic Compounds
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The reagents and chemicals for the targeted scheme were obtained from Merck (Burlington, MA, USA), Fischer (Waltham, MA, USA) and Acros Organics (Guglielmo Marconi, Verona, Veneto, Italy) and were used as supplied. All solvents were of analytical grade and were purified by distillation before use in the experimental protocols. FT-IR (Fourier transform infrared spectra) were estimated on the Bruker OPUS FT-IR spectrometer by attenuated total reflection (Diamond ATR) on solid films. Meanwhile, 1H NMR and 13C NMR were recorded via the Bruker DPX-400 and at 500 MHz, AV400, or AV(III)400 (Bruker, Zurich, Switzerland) machines using deuterated chloroform (CDCl3) and were employed to report chemical shifts in ppm. The ESI-HRMS data were recorded using the Bruker Micro TOF-ESI (Bruker Daltonics, Germany) positive targeted mode. The elemental analysis (CHN) was performed on the CE-440 Elemental Analyzer (Exeter Analytical (Coventry, UK) Ltd.). Melting points were estimated on the Gallenkamp instrument (Fisons, Uckfield, UK).
6
Multidomain Peptide Synthesis and Characterization
7
Characterization of C. vicina AMP Complex
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Natural compound containing C. vicina AMP complex was characterized by a combination of reversed phase HPLC, MS and bacterial growth inhibition assays. 1 mg of the lyophilized compound was dissolved in deionized water and applied to Shimadzu LC20 Prominence HPLC system equipped with analytical column C18 Vydac (4.6 х 250 mm, 5 μm, Grace), equilibrated with 0.05% TFA. The column was eluted with a linear gradient of acetonitrile (ACN) from 0 to 50% in acidified water (0.05% TFA) for 50 min [17 ]. Chromatographic fractions were automatically collected with 1 min intervals. The fractions’ optical densities were registered by means of a UV detector at two fixed wavelengths 214 and 280 nm. The fractions were lyophilized, dissolved in deionized water and tested against M. luteus A270 and E. coli D31 using the plate growth inhibition assay described below. Active antibacterial fractions were analyzed by MS (MicroTOF ESI, Bruker Daltonics) and experimentally determined masses were compared with the previously published characteristics of C. vicina individual AMPs [8 (link), 10 ]. The peptides were sequenced by Edman degradation method as described [10 ].
8
Synthesis of Ethyl 2-[2-(2-nitrobenzylidene)hydrazinyl]thiazole-4-carboxylate
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Ethyl 2-[2-(2-nitrobenzylidene)hydrazinyl]thiazole-4-carboxylate (1) was synthesized by the reaction of 1-(2-nitrobenzylidene)Thiosemicarbazide and ethyl bromopyruvate (Scheme 1 ). The pure reagent grade chemicals were purchased and used as such. Thiosemicarbazide, 4-nitrobenzaldehyde and ethyl bromopyruvate were purchased form Merck Germany. Pre-coated Silica 60 HF254 Aluminum sheets (Merck, Germany) were used to monitor the reaction with thin layer chromatography (TLC). The melting point determination, functional group identification, NMR chemicals shifts and single crystal analysis were carried out as reported elsewhere [25 ]. Dimethyl sulfoxide-deuterated (DMSO-d6) was used to record the proton and carbon NMR (nuclear magnetic resonance), using 300 and 75 MHz frequency, respectively. High resolution mass spectrometry (HRMS) was carried out on Bruker Micro TOF-ESI (time of flight-electrospray ionization) spectrometer, positive targeted mode.![]()
Synthesis of ethyl 2-[2-(2-nitrobenzylidene)hydrazinyl]thiazole-4-carboxylate
9
Identifying Metabolites Affected by Naringenin
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To identify molecular species in DOCplant that are affected by naringenin, we separated the ensemble of chemicals in the DOC chemicals using an HPLC (1200 HPLC, Agilent, Santa Clara, CA) and analyzed their mass using micrOTOF ESI (Bruker, Billerica, MA). Chemicals were bound to a Luna C18 reversed-phase column (Phenomenex Inc., Torrance, CA) by injecting 25 μl of DOC before or after the addition of naringenin or EDTA. The mobile phase consisted of a mixture of 0.1% formic acid in water (pump A) and 0.1% formic acid in acetonitrile (pump B) (49 ). The flow rate was 0.25 ml min−1 following the gradient program: 0 to 11 min at 80% A, 11 to 18 min at 30% A, and 18 to 25 min at 80% A. The MS was performed using ion positive mode to scan over the range of 100 to 2000 m/z. Data were analyzed using Bruker Compass Data Analysis 4.2. Graphs are shown as total ion chromatogram within the m/z scanning range. Retention time and mass spectrum fragmentation of naringenin and EDTA were compared using naringenin dissolved in ethanol and EDTA prepared in water, with the pH adjusted to 8.0 using 1 M sodium hydroxide.
10
Mass Spectrometry Analysis of Henna
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Mass spectroscopy of the methanolic extract of Henna was determined in methanol using MicroTof-ESI (Bruker Daltonics, Germany). Additionally, the fractionization and mass spectroscopy of the extract was carried out using 6110 HPLC-MS-ESI (Agilent Technologies, USA). In all cases, both positive and negative ionisation modes of the mass spectroscopy were acquired.
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