MS2 analyses of mono-deacetylated CSN-MN products (i.e. GlcNnGlcNAc, with n ≥ 1) were carried out by dissolving 5 µg of chitosan oligomers in 10 µl of 18O-labelled water and 0.1% formic acid. Samples were incubated overnight at 70 °C to exchange the hydroxyl group at the reducing end of oligomers with 18OH. Samples were freeze-dried and dissolved in 10 µl deionized water, and 1 µl of this solution was used for LC-MS using a Waters Acquity BEH Amide column (1.7 µm, 2.1 mm × 150 mm) in a Dionex Ultimate 3000 UHPLC coupled to an ESI-mass spectrometer (Amazon speed, Bruker Daltonics). MS2 was performed in positive mode and data were evaluated using the Bruker Compass Data Analysis program. The abundance of different mono- deacetylated chitosan oligomers was quantified based on the method described by Cord-Landwehr et al.40 . Briefly, oligomers were re-N-acetylated using [2H6] acetic anhydride and labelled at the reducing ends using H218O. To determine the abundance of different oligomer sequences, the largest b-ion and all y-ions of each precursor were considered. Scripts written in the Python programming language were used to evaluate the sequencing data41 .
Compass data analysis program
Manufactured by Bruker
Sourced in Germany
The Compass Data Analysis program is a software suite developed by Bruker to facilitate the processing and analysis of data obtained from various analytical instruments. The core function of the program is to provide users with tools for visualization, manipulation, and interpretation of data.
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Lab products found in correlation
Acquity beh amide column, by Waters Corporation (1 mentions)
Superdex 200 16 60 size exclusion column, by GE Healthcare (1 mentions)
Microtof q, by Bruker (1 mentions)
Orbitrap elite, by Thermo Fisher Scientific (1 mentions)
C18 ziptip, by Merck Group (1 mentions)
Peaks software, by Bioinformatics Solutions (1 mentions)
Microtof q spectrometer, by Bruker (1 mentions)
Tunemix mixture, by Bruker (1 mentions)
3 protocols using compass data analysis program
1
Deacetylated Chitosan Oligomers MS2 Analysis
2
Cyclic Product Purification and Characterization
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Full-length MSP2 (0.55 mg mL−1; 22 µM) was incubated with AEPs (0.066–0.68 µM) in non-reducing activity buffer (50 mM sodium acetate, 50 mM NaCl, 1 mM EDTA, pH 5) for 30–60 min. The reaction was monitored by SDS PAGE followed by Coomassie blue staining. To confirm the presence of cyclic product, the processing product was gel extracted and subjected to trypsin digest followed by tandem MS/MS analysis using an Orbitrap Elite (Thermo Scientific). The resulting spectra were analysed using PEAKS software (version 8.5; Bioinformatics Solutions, Inc.).
For purification of the cyclic product, TFA was added to a final concentration of 0.1% and the reaction mix was loaded onto a Superdex 200 16/60 size exclusion column (GE Healthcare) equilibrated in PBS at a flow rate of 0.5 ml min−1. Fractions positive for cyclic product by SDS PAGE followed by Coomassie blue staining were pooled and analysed further by ESI MS. Fractions were desalted using C18 zip tips (Millipore) according to the manufacturer’s instructions and eluted in 4 µL 75% (v/v) methanol, 1% (v/v) formic acid. 96 µl of 50% (v/v) methanol, 1% (v/v) formic acid was added to the desalted sample. The sample was then injected into a MicroTOF Q (Bruker) and data was collected in positive ionisation mode. The mass was determined by charge deconvolution using the Compass DataAnalysis program (Bruker).
For purification of the cyclic product, TFA was added to a final concentration of 0.1% and the reaction mix was loaded onto a Superdex 200 16/60 size exclusion column (GE Healthcare) equilibrated in PBS at a flow rate of 0.5 ml min−1. Fractions positive for cyclic product by SDS PAGE followed by Coomassie blue staining were pooled and analysed further by ESI MS. Fractions were desalted using C18 zip tips (Millipore) according to the manufacturer’s instructions and eluted in 4 µL 75% (v/v) methanol, 1% (v/v) formic acid. 96 µl of 50% (v/v) methanol, 1% (v/v) formic acid was added to the desalted sample. The sample was then injected into a MicroTOF Q (Bruker) and data was collected in positive ionisation mode. The mass was determined by charge deconvolution using the Compass DataAnalysis program (Bruker).
3
Mass Spectrometry Analysis of Metal Complexes
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All MS experiments were performed on a Bruker microTOF-Q spectrometer (Bruker Daltonik, Bremen, Germany), equipped with an Apollo II electrospray ionization source with an ion funnel. The instrument parameters were: scan range m/z 250-2000, dry gasnitrogen, temperature 200 °C, ion source voltage 4500 V, collision energy 10 eV, analyte aqueous solutions (70 μL). Solutions were introduced at a flow rate of 3 μL min -1 . The Bruker microTOF-Q spectrometer was calibrated with the Tunemix™ mixture (Bruker Daltonik, Germany) in the quadratic regression mode. The mass spectrometer operated in the positive ion mode. Each spectrum was obtained with more than 100 individual scans. The overall charge of the analysed ions was calculated on the basis of the distance between isotopic peaks. The formulae of the complexes were determined by application of the Compass Data Analysis program (Bruker Daltonik, Germany). 22 The water and D 2 O solutions of the ligand (1 × 10 -4 M) with FeCl 3 and AlCl 3 salts (1 : 3 metal to ligand molar ratio) and CuCl 2 , ZnCl 2 (1 : 2 metal to ligand molar ratio) were incubated for 15 days at room temperature before the measurements. Solutions at variable pH and pD values were obtained by adding triethylamine and HCl (water solution), or NaOD and DCl (D 2 O solutions). pH and pD were measured on a previously calibrated pHmeter.
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