The detailed procedures were reported previously.15 (link) Briefly, the N-glycans were released from cell membrane using PNGase F after overnight incubation at 37 °C. The released N-glycans were purified using porous graphitic carbon (PGC) SPE plates, the glycan samples were analyzed with an Agilent 6520 Accurate Mass Q-TOF LC/MS equipped with a PGC nano-chip (Agilent, CA), and the results were extracted with the MassHunter Qualitative Analysis B08 software (Agilent, CA). For glycoproteomic analysis, glycopeptides after trypsin digestion were enriched by solid-phase extraction using iSPE®-HILIC cartridges (Nest Group, MA). The enriched glycopeptides were characterized using a UltiMate™ WPS-3000RS nanoLC system coupled with an Orbitrap Fusion Lumos (ThermoFisher Scientific), and an in-house human N-glycan database was applied to the raw results using the Byonic software (Protein Metrics, CA).
Masshunter qualitative analysis b08
Manufactured by Agilent Technologies
MassHunter Qualitative Analysis B08 software is a data analysis application developed by Agilent Technologies. It is designed to process and interpret data generated by mass spectrometry instruments.
Automatically generated - may contain errors
Lab products found in correlation
Pgc nano chip, by Agilent Technologies (3 mentions)
Agilent 6520 accurate mass q tof lc ms, by Agilent Technologies (2 mentions)
Orbitrap fusion lumos, by Thermo Fisher Scientific (1 mentions)
Ultimate wps 3000rs nanolc system, by Thermo Fisher Scientific (1 mentions)
Byonic software, by Protein Metrics (1 mentions)
6520 accurate mass q tof lc ms, by Agilent Technologies (1 mentions)
Mass hunter quantitative analysis b 08, by Agilent Technologies (1 mentions)
Agilent 1290 uplc system, by Agilent Technologies (1 mentions)
Agilent 6550 q tof mass spectrometer, by Agilent Technologies (1 mentions)
6 protocols using masshunter qualitative analysis b08
1
Glycoproteomic Analysis of N-Glycans
2
N-Glycan Analysis by Mass Spectrometry
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The cell membrane fractions were resuspended with 100 μL of 5 mM DTT in 100 mM ammonium bicarbonate. The mixture was heated in boiling water for 3 minutes. The cleavage of N-glycans was performed by adding 2 μL of PNGase F followed by incubation in a 37 °C water bath overnight. The released N-glycans were separated using centrifugation at 200 000×g for 30 minutes, and the supernatant was purified using porous graphitic carbon (PGC) on an SPE plate. The glycan samples were dried and reconstituted in 30 μL nanopure water. 5 μL of the sample was injected and analyzed with an Agilent 6520 Accurate Mass Q-TOF LC/MS equipped with a PGC nano-chip (Agilent, CA). A binary gradient using solvent A with 3% (v/v) ACN and 0.1% (v/v) formic acid in water and solvent B with 90% (v/v) ACN and 1% (v/v) formic acid in water was applied to separate N-glycans at a flow rate of 300 nL min−1. The resulting chromatographs of glycans were extracted with the MassHunter Qualitative Analysis B08 software (Agilent, CA). N-glycan compounds were identified with an in-house library that contains the accurate mass and formula of human N-glycans, and the N-glycan structures were confirmed through tandem MS fragmentation.
3
N-Glycan Purification and Analysis
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The cell membrane fractions were resuspended with 100 μL of 5 mM DTT in 100 mM ammonium bicarbonate. The mixture was heated in boiling water for 3 minutes. The cleavage of N-glycans was performed by adding 2 μL of PNGase F followed by incubation in a 37 °C water bath overnight. The released N-glycans were separated using 200 000×g for 30 minutes, and the supernatant was purified using porous graphitic carbon (PGC) on an SPE plate. The glycan samples were dried and reconstituted in 30 μL of nanopure water. The sample (5 μL) was injected and analyzed with an Agilent 6520 Accurate Mass Q-TOF LC/MS equipped with a PGC nano-chip (Agilent, CA). A binary gradient using solvent A with 3% (v/v) ACN and 0.1% (v/v) formic acid in water and solvent B with 90% (v/v) ACN and 1% (v/v) formic acid in water was applied to separate N-glycans at a 300 nL min−1 flow rate. The resulting chromatograms of glycans were extracted with the MassHunter Qualitative Analysis B08 software (Agilent, CA). N-Glycan compounds were identified using GlycoNote (https://github.com/MingqiLiu/GlycoNote ), which contains the accurate mass and formula of human N-glycans, and the N-glycan structures were confirmed through tandem MS fragmentation.
4
Agilent MassHunter Data Processing Protocol
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Agilent MassHunter Qualitative Analysis B.08.00 and Agilent MassHunter Quantitative Analysis B.08.00 were used for processing of obtained raw data. For semi-quantitative analysis a minimal signal to noise ratio of 1:5 and a retention time deviation of 0.4 min were set as cut-off for peak integration. Quantifier MRM transitions were used for peak integration. Peak areas were divided by the internal standard isoguanosine. Blanks were subtracted and peak areas were further normalised by division of total peak areas of the chromatogram. Finally each variable was mean-centred and divided by the range of each variable, called range scaling33 (link)–38 (link).
5
UPLC-QTOF-MS Analysis of Compounds
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Liquid chromatography was performed on an Agilent 1290 UPLC system (Agilent Technologies, USA) equipped with a binary pump, an auto-plate sampler, a thermostatically controlled column compartment and a diode array detection system. Detection was performed by an Agilent 6550 Q-TOF mass spectrometer (Agilent Technologies, USA). Chromatographic separation was conducted on an Agilent extend C18 (2.1 × 50 mm, 1.7 um). The mobile phase was composed of 0.1% formic acid solution (A) and acetonitrile solution (B), with a gradient elution procedure as follows: 0–1 min, 90% A, 10% B; 1–3 min, 10% A, 90% B; 1–3 min, 10% A, 90% B; 12–13 min, 90% A, 10% B. The flow rate was 0.3 mL/min, and the injection volume was 10 mL.
The MS spectra were acquired in both positive and negative ion modes to provide complementary information for structural identification. The acquisition parameters for Q-TOF mass spectra were as follows: drying gas temperature, 150 °C; drying gas flow rate, 15.0 L/min; sheath gas temperature, 350 °C; sheath gas flow rate, 12.0 L/min; capillary voltage, 4500 V (positive)/−4000 V (negative); the mass range was recorded from 50 to 1000 m/z. Agilent Mass Hunter Qualitative Analysis B.08.00 was used for data collection and processing.
The MS spectra were acquired in both positive and negative ion modes to provide complementary information for structural identification. The acquisition parameters for Q-TOF mass spectra were as follows: drying gas temperature, 150 °C; drying gas flow rate, 15.0 L/min; sheath gas temperature, 350 °C; sheath gas flow rate, 12.0 L/min; capillary voltage, 4500 V (positive)/−4000 V (negative); the mass range was recorded from 50 to 1000 m/z. Agilent Mass Hunter Qualitative Analysis B.08.00 was used for data collection and processing.
6
Metabolomic Profiling and Pathway Analysis
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The identification of metabolites was carried out using the METLIN_Metabolites Database on Agilent MassHunter Qualitative Analysis B.08.00 software and the lists for data analysis were generated with compounds present in all the replicates of each extract. Putative assignments for each compound were made based on their accurate mass. Additionally the Dictionary of Natural Products, PubChem, (http://pubchem.ncbi.nlm.nih.gov/ , accessed on 11 October 2020), LIPID MAPS (http://www.lipidmaps.org/tools , accessed on 11 October 2020), and Human Metabolome Database (HMDB) (http://www.hmdb.ca , accessed on 11 October 2020) were consulted. Principal component analysis (PCA), Venn diagram, and fold change analysis (cut off 2.0) were carried out for UHPLC-QTOF-MS/MS data on Mass Profiler Professional software. PCA presents the average of replicates by each extract. For all statistical tests performed, ANOVA with cut-off p < 0.05 was taken as significant. The metabolomics pathway analysis and the network topological analysis were performed with MetaboAnalyst (http://www.metaboanalyst.ca/ , accessed on 3 February 2021) and STITCH (http://stitch.embl.de/ , accessed on 3 February 2021). The metabolite-gene-disease interaction network was selected within the MetPA module, through the integration of network topological analysis, interactive network exploration, and functional enrichment analysis.
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