All synthetic peptides were purchased from GME biochemicals (Shanghai, China) at 95% purity. Ac-YSNGF, Ac-YSDGF, Ac-YHNEF, Ac-YHDEF and Ac-YHNPF (1mg/mL) were incubated at pH 5.0 (100mM citric acid), pH 6.7 or pH 7.4 (100mM sodium phosphate) at 60°C. Aliquots were taken at intervals and injected onto a C18 RP HPLC column (Aeris, 2.6μ, XB-C18, 100mm x 2.1mm, Phenomenex). In these and the experiments below, a 50-minute gradient was performed at a flow rate of 0.2mL/min, consisting of the following: 0-5 min, 2% CH3CN; 5-15 min, 20% CH3CN, 15-20min 40% CH3CN, 25-35min 80% CH3CN, 35-50 2% CH3CN. Peptide cleavage products were detected at 216nm and 280nm, collected, lyophilised and their identities confirmed by tandem mass spectrometry using a Thermo LTQ mass spectrometer (Thermo Scientific). Since there was no difference in the MS/MS spectra of C-terminal Asn and Asp-amides, their identity was confirmed by the elution times of synthetic standards. The percentage of cleavage of each peptide was determined by the peak area of cleavage products compared with that of the original peptide at 216nm and 280nm. All experiments were run in triplicate.
Thermo ltq mass spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Thermo LTQ mass spectrometer is a high-performance liquid chromatography-mass spectrometry (LC-MS) system. It is designed to provide accurate and sensitive analysis of complex samples. The instrument utilizes linear ion trap technology to efficiently capture, isolate, and detect ions for mass analysis.
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Lab products found in correlation
Thermo ltq orbitrap xl mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Ft ir nir spectrometer, by PerkinElmer (1 mentions)
Autopol 4 automatic polarimeter, by Rudolph Research Analytical (1 mentions)
Arx 600 spectrometer, by Bruker (1 mentions)
J 815 spectropolarimeter, by Jasco (1 mentions)
Lithium hydroxide, by Merck Group (1 mentions)
Potassium persulfate, by Merck Group (1 mentions)
2 acrylamido 2 methylpropane sulfonic acid, by Merck Group (1 mentions)
Phosphate buffered saline pbs, by Corning (1 mentions)
Mueller hinton broth (mhb), by BD (1 mentions)
N isopropylacrylamide, by Merck Group (1 mentions)
Acrylic acid, by Merck Group (1 mentions)
Resazurin sodium salt, by Merck Group (1 mentions)
N n methylenebisacrylamide, by Merck Group (1 mentions)
Methyl acrylate, by Merck Group (1 mentions)
Protease complete, by Roche (1 mentions)
9 protocols using thermo ltq mass spectrometer
1
Peptide Stability at Different pH
2
Quantification of Lysophosphatidylcholines by ESI-MS
3
Mass Spectrometry-Based Single-Cell Analysis
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Except for the single cell analysis, all other experiments were carried out using a Thermo LTQ mass spectrometer (Thermo Scientific, San Jose CA). The instrumental parameters were as follows: capillary temperature = 275 °C and max injection time = 200 ms. The single cell amount analysis was carried out using a Thermo QE-Orbitrap mass spectrometer (Thermo Scientific, San Jose CA). The capillary temperature was 320 °C. The analytical method was full MS, followed by data dependent MS2. The method duration was 5 min. Full MS: resolution = 70 000, AGC = 3 × 106, max injection time = 100 ms, and scan range = 134 to 2000. The ddMS2 settings: resolution = 35 000, AGC = 1 × 105, ion injection time = 100 ms, loop count = 10, NCE = 30, underfill ratio = 0.1%, and dynamic exclusion = 300 s.
4
Mass Spectrometry Protocol for DESI Analysis
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Example 1
Experiments were carried out using a custom built miniature mass spectrometer (Mini 10; Gao et al., Anal. Chem. 2006, 78, 5994-6002) or a Thermo LTQ mass spectrometer (Thermo Scientific, San Jose, Calif.). Capillary temperature: 150° C.; capillary voltage: 15 V; tube lens voltage: 240 V. A custom power supply provided a pulsed output of 50-5,000 Hz and 0-8 kV. DESI (Takats et al., Science 2004, 306, 471-473) conditions were: nitrogen gas 150 psi, a metal tube (id 250 μm, 5 cm long) serves as outer electrode, an inner silica capillary serves as the spray emitter (id 50 μm), angle of DESI sprayer to substrate set at 40°, distance between spray tip and sample set at 2 mm, distance between sample and MS inlet, 3 mm; the spray solution was MeOH/water (v:v=1:1), Commercial silica nanoelectrospray tips of 20 μm were obtained from New Objective (Woburn, Mass., USA).
5
Miniature Mass Spectrometer Protocol
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Example 1
Experiments were carried out using a custom built miniature mass spectrometer (Mini 10; Gao et al., Anal. Chem. 2006, 78, 5994-6002) or a Thermo LTQ mass spectrometer (Thermo Scientific, San Jose, Calif.). Capillary temperature: 150° C.; capillary voltage: 15 V; tube lens voltage: 240 V. A custom power supply provided a pulsed output of 50-5,000 Hz and 0-8 kV. DESI (Takats et al., Science 2004, 306, 471-473) conditions were: nitrogen gas 150 psi, a metal tube (id 250 μm, 5 cm long) serves as outer electrode, an inner silica capillary serves as the spray emitter (id 50 μm), angle of DESI sprayer to substrate set at 40°, distance between spray tip and sample set at 2 mm, distance between sample and MS inlet, 3 mm; the spray solution was MeOH/water (v:v=1:1), Commercial silica nanoelectrospray tips of 20 μm were obtained from New Objective (Woburn, Mass., USA).
6
Miniature Mass Spectrometer Protocol
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Example 1
Experiments were carried out using a custom built miniature mass spectrometer (Mini 10; Gao et al., Anal. Chem. 2006, 78, 5994-6002) or a Thermo LTQ mass spectrometer (Thermo Scientific, San Jose, Calif.). Capillary temperature: 150° C.; capillary voltage: 15 V; tube lens voltage: 240 V. A custom power supply provided a pulsed output of 50-5,000 Hz and 0-8 kV. DESI (Takats et al., Science 2004, 306, 471-473) conditions were: nitrogen gas 150 psi, a metal tube (id 250 μm, 5 cm long) serves as outer electrode, an inner silica capillary serves as the spray emitter (id 50 μm), angle of DESI sprayer to substrate set at 40°, distance between spray tip and sample set at 2 mm, distance between sample and MS inlet, 3 mm; the spray solution was MeOH/water (v:v=1:1), Commercial silica nanoelectrospray tips of 20 μm were obtained from New Objective (Woburn, Mass., USA).
7
Characterization of Organic Compounds
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Optical rotations were recorded on an Autopol IV automatic polarimeter with a 10 cm glass microcell at 25 °C (Rudolph Research Analytical, NJ, USA). The CD spectra were measured on a JASCO J-815 spectropolarimeter using CH3OH as the solvent at room temperature (Jasco Corporation, Tokyo, Japan). UV spectra were obtained in CH3OH on a Persee TU-1901 UV-vis spectrometer (Beijing Purkinje General Instrument Co., Ltd, Beijing, China). IR spectra were acquired on a PerkinElmer FT-IR/NIR spectrometer (PerkinElmer, Waltham, MA, USA). 1D and 2D NMR spectra were performed at 600 MHz for 1H NMR and 150 MHz for 13C NMR on Bruker ARX-600 spectrometer using solvent signals as the internal standard (Bruker, Switzerland). Chemical shifts (δ) are given in ppm, and coupling constants (J) are given in hertz (Hz). ESIMS data were recorded on a Thermo LTQ mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). HRESIMS data were measured using a Thermo LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Column chromatography (CC) were carried out with silica gel (200–300 mesh, Qingdao Marine Chemical Inc. Qingdao, PR China). Analytical TLC was carried out on pre-coated silica gel GF254 plates (Qingdao Marine Chemical Industry, Qingdao, China), and spots were visualized under UV light or by spraying with 10% H2SO4 in 90% EtOH followed by heating at 120 °C.
8
Synthesis and Characterization of Antimicrobial Peptides
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The peptides used in these studies were custom synthesized by ChinaPeptides Company (Shanghai, China) and had purities of ≥ 95%, based on chromatographic analysis of the purified peptides. Synthetic peptides were verified on a Thermo LTQ mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The broad-spectrum protease inhibitor cocktail Protease cOmplete was purchased from Roche Diagnostic, Corp. (Indianapolis, IN). Resazurin, sodium salt is purchased from Sigma-Aldrich (St. Louis, MO). N-Isopropylacrylamide (NIPAm), N, N′-Methylenebisacrylamide (BIS), Acrylic acid (AAc), 2-Acrylamido-2-methylpropane sulfonic acid (AMPS), Methyl Acrylate (MA), Lithium hydroxide (LiOH) and potassium persulfate (KPS) are all purchased from Sigma-Aldrich (St. Louis, MO). Mueller Hinton Broth (MHB) was purchased from Becton Dickinson and Company (Sparks, MD). Phosphate buffered saline (PBS) was purchased from Corning-cellgro (Manassas, VA). Commercial alligator plasma was purchased from Bioreclamation (Westburg, NY). Alligator blood was acquired from St. Augustine’s Alligator Farm (St. Augustine, FL). All experiments involving alligators were carried out with compliance with relevant guidelines, using protocols approved by the GMU IACUC.
9
Investigating Peptide Stability in pH Solutions
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