Mass spectrometry analysis was performed on a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF–MS) AXIMA CFR (Kratos Analytical). α-Cyano-4-hydroxycinnamic acid (CHCA) was used as matrix. The specific parameters were as follows: the ion acceleration voltage was 20 kV, the accumulating time of single scanning was 50 s, polypeptide mass standard (Kratos Analytical) serving as external standard.
Axima cfr
Manufactured by Shimadzu
Sourced in United Kingdom, Japan
The AXIMA-CFR is a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer designed for high-performance analysis of biomolecules. It provides accurate mass measurements and detailed structural information for a wide range of sample types, including proteins, peptides, lipids, and oligonucleotides.
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Lab products found in correlation
Polypeptide mass standard, by Shimadzu (2 mentions)
11 elite, by Harvard Apparatus (2 mentions)
Axima resonance, by Shimadzu (1 mentions)
V 670 spectrophotometer, by Jasco (1 mentions)
2 5 dihydroxybenzoic acid, by Merck Group (1 mentions)
Model 433a, by Thermo Fisher Scientific (1 mentions)
V 650, by Jasco (1 mentions)
14 protocols using axima cfr
1
MALDI-TOF Mass Spectrometry Analysis
2
Polypeptide Mass Characterization by MALDI-TOF
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The purified peptide was subjected to amino acid sequencing by Edman degradation on an Applied Biosystems pulsed liquid-phase sequencer, model 491. The Cys sulfhydryl group was alkylated by reaction with 4-vinyl pyridine according to the method described by Friedman et al. [7] . The actual molecular mass of the peptide was measured by a Matrix-Assisted Laser Desorption Ionization Time-Of-Flight mass spectrometer (MALDI-TOF-MS, AXIMA CFR (Kratos Analytical)) in positive ion and linear mode. α-Cyano-4-hydroxycinnamic acid (CHCA) was used as matrix. The specific operating parameters were as follows: the ion acceleration voltage was 20 kV; the accumulating time of single scanning was 50 s. The polypeptide mass standard (Kratos Analytical) was used as the external standard.
3
Solid-Phase Synthesis of Peptides
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Peptides, herein termed CP2a ( ) and CP2b ( ), were synthesized by the solid phase method using Fmoc (9-fluorenyl-methoxycarbonyl) chemistry. Their specific length was chosen to ensure that a completely unfolded linear peptide can fit inside the ~ 10 nm thick α-HL pore (vide infra).
Rink Amide 4-methyl benzhydrylamine (MBHA) resin (0.30 mmol/g) was used as the support to obtain a C-terminal amidated peptide. The coupling of Fmoc-L-amino acids was performed with O-Benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate(HBTU). Amino acid side chains were protected with tert-butyl and tert-butyloxycarbonyl. Deprotection and cleavage from the resin were carried out using a mixture of trifluoroacetic acid, phenol, water, thioanisole, 1,2-ethandithiol (82.5, 5.0, 5.0, 5.0, 2.5, v/v) for 3 h at room temperature. The crude peptide was then repeatedly washed with diethylether, dried in vacuum, and purified using a preparative reversed-phase HPLC (RP-HPLC) on a Shimadzu 5-μm Shimpak ODS C18 column (20 × 250 mm). Purity of the peptide was checked by analytical RP-HPLC on a Shimpak ODS C18 column (4.6 × 250 mm). The molecular masses of the synthetic peptides were determined using the matrix-assisted laser desorption ionization MALDI-TOF mass spectrometer (Axima CFR, Kratos Analytical, Manchester, UK).
Rink Amide 4-methyl benzhydrylamine (MBHA) resin (0.30 mmol/g) was used as the support to obtain a C-terminal amidated peptide. The coupling of Fmoc-L-amino acids was performed with O-Benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate(HBTU). Amino acid side chains were protected with tert-butyl and tert-butyloxycarbonyl. Deprotection and cleavage from the resin were carried out using a mixture of trifluoroacetic acid, phenol, water, thioanisole, 1,2-ethandithiol (82.5, 5.0, 5.0, 5.0, 2.5, v/v) for 3 h at room temperature. The crude peptide was then repeatedly washed with diethylether, dried in vacuum, and purified using a preparative reversed-phase HPLC (RP-HPLC) on a Shimadzu 5-μm Shimpak ODS C18 column (20 × 250 mm). Purity of the peptide was checked by analytical RP-HPLC on a Shimpak ODS C18 column (4.6 × 250 mm). The molecular masses of the synthetic peptides were determined using the matrix-assisted laser desorption ionization MALDI-TOF mass spectrometer (Axima CFR, Kratos Analytical, Manchester, UK).
4
AXIMA Mass Spectrometry Protocols
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AXIMA Resonance and AXIMA CFR mass spectrometers, both from Kratos Analytical Ltd. (Manchester, UK), coupled with a quadrupole ion trap and time-of-flight detection, were used to record mass spectra in both positive and negative ion modes. Both instruments were equipped with a nitrogen laser (337 nm), and the laser repetition rate was set to 5 Hz with a pulse time width of 3 ns.
5
MALDI-TOF Mass Spectra Acquisition
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MALDI-TOF mass spectra were acquired on a time-of-flight mass spectrometer AXIMA-CFR (Shimadzu, Kyoto, Japan) equipped with a nitrogen laser (337 nm wavelength) operating at a pulse rate of 10 Hz. The pulse width of the laser was 4 ns. The laser spot size on the target substrate was ca. 100 μm in diameter. The ions generated by MALDI were accelerated using 20 kV with delayed extraction. The analyzer was operated in linear mode and the ions were detected using a secondary electron multiplier. A total of 500 shots were accumulated for each mass spectrum acquisition. The reproducibility of all ISD spectra were confirmed by the peak intensity patterns for several runs using the raster function installed on the AXIMA-CFR mass spectrometer.
6
Comprehensive Spectroscopic Characterization of Novel Compounds
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UV–vis
optical spectra of
the products were measured by a Jasco V-670 spectrophotometer using
2 and 10 mm cuvettes. Electrospray ionization (ESI) mass spectra were
recorded on a Bruker compact ESI-Q-TOF-MS. Matrix-assisted laser desorption/ionization
(MALDI) mass spectra were recorded by a Shimadzu AXIMA-CFR. DCTB was
used as a matrix. The sample (0.1 mg) and DCTB (1.5 mg) were dissolved
in toluene (20 μL), and a few microliters of the mixed solution
were spotted on the MALDI plate. Gel permeation chromatography was
conducted using a YMC-GPC T30000 column with toluene as an eluent.
X-band CW-EPR measurement was carried out using a Bruker E500 spectrometer
with a 4He flow cryostat. The DCM solution was placed in
a quartz tube (4 mmφ) and rapidly frozen by immersing in liquid
N2. The frozen solution was quickly transferred to the
precooled cryostat, and measurement was conducted at several temperatures.
The spectrum shown in this paper was recorded by using the following
parameters: microwave frequency = 9.67 GHz, microwave power = 0.1
mW, and modulation amplitude = 0.500 mT. The microwave power dependency
indicated that the condition was far less than saturation (data not
shown). The EPR spectrum was simulated using the EasySpin toolbox
in MATLAB 9.6.0.5. The range of 150–550 mT was used for fitting.
optical spectra of
the products were measured by a Jasco V-670 spectrophotometer using
2 and 10 mm cuvettes. Electrospray ionization (ESI) mass spectra were
recorded on a Bruker compact ESI-Q-TOF-MS. Matrix-assisted laser desorption/ionization
(MALDI) mass spectra were recorded by a Shimadzu AXIMA-CFR. DCTB was
used as a matrix. The sample (0.1 mg) and DCTB (1.5 mg) were dissolved
in toluene (20 μL), and a few microliters of the mixed solution
were spotted on the MALDI plate. Gel permeation chromatography was
conducted using a YMC-GPC T30000 column with toluene as an eluent.
X-band CW-EPR measurement was carried out using a Bruker E500 spectrometer
with a 4He flow cryostat. The DCM solution was placed in
a quartz tube (4 mmφ) and rapidly frozen by immersing in liquid
N2. The frozen solution was quickly transferred to the
precooled cryostat, and measurement was conducted at several temperatures.
The spectrum shown in this paper was recorded by using the following
parameters: microwave frequency = 9.67 GHz, microwave power = 0.1
mW, and modulation amplitude = 0.500 mT. The microwave power dependency
indicated that the condition was far less than saturation (data not
shown). The EPR spectrum was simulated using the EasySpin toolbox
in MATLAB 9.6.0.5. The range of 150–550 mT was used for fitting.
7
MALDI-TOF/MS Analysis of PA-Oligosaccharides
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PA-oligosaccharides were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOF/MS). The matrix solution was prepared as follows: 10 mg of 2,5-Dihydroxybenzoic acid (Sigma) was dissolved in 1∶1 (v/v) acetonitrile/water (1 mL). Stock solutions of PA-glycans were prepared by dissolving them in pure water. One microliter of a sample solution was mixed on the target spot of a plate with 1 µL matrix solution and then allowed to air-dry. MALDI-TOF/MS data were acquired in the positive mode on an AXIMA-CFR (Shimadzu) operated in linear mode.
8
Agaphelin (1-58) Peptide Folding
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Linear, purified peptide Agaphelin (1–58) was dissolved in 6 M guanidin•HCl/200 mM PBS at a concentration of 2 mmol, and the solution was introduced by a Harvard Apparatus “Elite 11” through a syringe, to a 30 times larger volume of the stirred solution of degassed, 50 mM Tris/1 mM EDTA, pH∼8, containing reduced glutathione and oxidized glutathione at concentrations of 1.6 mM and 0.2 mM, respectively. The progress of folding was monitored by HPLC using a gradient of acetonitrile/water with UV monitoring at 215 nm. After 3 h, the reaction mixture was acidified with 2% trifluoroacetic acid to pH 5. The folded peptide was isolated by preparative reverse-phase HPLC and its purity checked by HPLC analyses and mass spectrometry using a matrix-assisted laser desorption ionization time-of-flight mass spectrometer Axima CFR+ (Shimadzu Scientific Instruments). Pure fractions were combined, frozen, and lyophilized to afford pure, folded peptide. Agaphelin molecular mass is 6273 Da (58 amino acids, mature form) with an estimated pI 5.09. Extinction coefficient at 280 nm is 3355; A280 nm/cm 0.1% (1 mg/ml), 0.535. Agaphelin was diluted in PBS (1–1.5 mM) and frozen at −80°C.
9
Quantification of Glycine Betaine and Choline
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Glycine betaine (GB) and choline were extracted from the control and E. coli cells expressing OsSHMT. E. coli cells were grown in liquid LB media containing various concentrations of NaCl (0, 100, 300, and 500 mM) in three replicates. Log phase cultures were taken for GB and choline extraction using KI-I2 method as described (Hibino et al., 2002 (link)). GB and choline were then quantified on a Time-of-flight mass spectrometer (AXIMA CFR, Shimadzu/Kratos, Japan) with d9-choline and d11-betaine, respectively, as an internal standard.
10
MALDI-TOF Mass Spectra Acquisition
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MALDI mass spectra were acquired on a time-of-flight mass spectrometer AXIMA-CFR (Shimadzu, Kyoto, Japan) equipped with a nitrogen laser (337 nm wavelength) operating at a pulse rate of 10 Hz. The pulse width of the laser was 4 ns. The laser spot size on the target substrate was ca. 100 μm in diameter. The ions generated by MALDI were accelerated using 20 kV with delayed extraction. The analyzer was operated in the linear and reflector mode and the ions were detected using a secondary electron multiplier. A total of 500 shots were accumulated for each mass spectrum acquisition.
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