Raw data from Masslynx 4.2 (Masslynx 4.2, Waters) software were transformed into.cdf format using the Masslynx Databridge tool. To process the data, R software v. 4.0.3 was used to separately analyze ESI + and ESI- signals. Signal corrections were obtained using the XCSM algorithm4 for R. The amount of each compound was determined from the normalized peak area units relative to the dry weight of each sample. Signals of different treatments were compared using the Kruskal–Wallis test (P < 0.05) following adduct and isotope correction. Mar-Vis Suit 2.0 was used to obtain isotope corrections, clustering, heatmaps (Mar-Vis Cluster), and pathways (Mar-Vis pathway) with different changes within treatments. MetaboAnalyst 4.0 was used for principal component analysis (PCA) applying normalization by median followed by cube root transformation and Pareto scaling. Raw data are available in Supplementary Table 2 .
Masslynx 4
Manufactured by Waters Corporation
Sourced in United States, United Kingdom, Spain, Ireland, Poland
MassLynx 4.1 is a software package designed to control and acquire data from mass spectrometry instruments. It provides a comprehensive interface for instrument operation, data acquisition, and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Acquity uplc system, by Waters Corporation (47 mentions)
Acquity uplc, by Waters Corporation (22 mentions)
Beh c18 column, by Waters Corporation (20 mentions)
Synapt g2 si mass spectrometer, by Waters Corporation (16 mentions)
Acquity uplc beh c18 column, by Waters Corporation (15 mentions)
Acquity ultra performance lc system, by Waters Corporation (15 mentions)
Acquity beh c18 column, by Waters Corporation (14 mentions)
Driftscope 2, by Waters Corporation (11 mentions)
Q tof premier, by Waters Corporation (11 mentions)
Xevo tq s mass spectrometer, by Waters Corporation (10 mentions)
Acquity uplc 1 class system, by Waters Corporation (9 mentions)
Acquity uplc h class, by Waters Corporation (9 mentions)
Acquity uplc 1 class, by Waters Corporation (9 mentions)
Triversa nanomate, by Advion (8 mentions)
Xevo tq xs, by Waters Corporation (7 mentions)
Acquity uplc beh shield rp18 column, by Waters Corporation (7 mentions)
Xevo tq s triple quadrupole mass spectrometer, by Waters Corporation (7 mentions)
Synapt g2 hdms, by Waters Corporation (7 mentions)
Xevo tq triple quadrupole mass spectrometer, by Waters Corporation (7 mentions)
Xcalibur 2, by Thermo Fisher Scientific (6 mentions)
586 protocols using masslynx 4
1
Metabolomic Workflow for Compound Quantification
2
Intact Protein Analysis by LC-MS
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Liquid chromatography–mass spectrometry (LC-MS) on intact protein samples was performed using an ACQUITY UPLC H-Class coupled in line to a ESI-Q-TOF Synapt G2-Si (Waters) using a Restek Ultra C4 5 μm (#9102551) reverse-phase column. The column was operated at 25 °C with the flow rate of 0.4 ml/min. Chromatography was performed with a linear gradient between mobile phases A and B, with Buffer A consisting of aqueous 0.1% formic acid and Buffer B consisting of 100% acetonitrile, 0.1% formic acid. The elution was carried out using a linear gradient from 5% to 100% B at a flow rate of 0.4 ml/min for 7 min. Protein samples were injected at a concentration of 0.1 mg/ml. Mass spectrometry was performed using a LockSpray ESI (Waters) ionization source with [Glu1]-fibrinopeptide B as internal lock mass calibrant. The instrument was operated using MassLynx 4.1 (Waters). MS data were collected in positive ion, MS continuum, resolution mode with an m/z range of 300 to 5000. The intact protein molecular weight of each sample was deconvoluted from multiple charge states using the MaxEnt3 function of MassLynx 4.2 (Waters).
3
Intact Protein Mass Spectrometry Analysis
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Protein intact masses were determined by LC-MS on a Synapt G2 mass spectrometer coupled to an Acquity UPLC system (Waters, Milford, MA, USA). 50–100 pmol of protein were injected onto an Aeris WIDEPORE 3.6μ C4 column (Phenomenex, UK) and eluted with a 10–90% acetonitrile gradient over 13 min (0.4 ml/min). The spectrometer was controlled by the Masslynx 4.1 software (Waters) and operated in positive MS-TOF and resolution mode with capillary voltage of 2 kV, cone voltage, 40 V. Leu-enkephalin peptide (2 ng/ml, Waters) was infused at 10 µl/min as a lock mass and measured every 30 s. Spectra were generated in Masslynx 4.1 by combining scans and deconvoluted using the MaxEnt1 tool (Waters).
4
HRESIMS Protocol for Compound Identification
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For HRESIMS, the samples were dissolved into water
+ 0.1% FA/methanol (1:1) and infused into a Synapt G2-Si mass spectrometer
(Waters, Manchester, U.K.) at 10 μL/min using a Harvard Apparatus
syringe pump. The mass spectrometer was controlled by Masslynx 4.1
software (Waters). It was operated in resolution and positive ion
mode and calibrated using sodium iodide. The sample was analyzed for
1 min with a 1 s MS scan time over the m/z range 50–1200 with 2.0 kV capillary voltage, 40
V cone voltage, and 120 °C cone temperature. Leu-enkephalin peptide
(1 ng/μL, Waters) was infused at 10 μL/min as a lock mass
(m/z 556.2766) and measured every
10 s. Spectra were generated in Masslynx 4.1 by combining multiple
scans, and peaks were centered using automatic peak detection with
lock mass correction.
+ 0.1% FA/methanol (1:1) and infused into a Synapt G2-Si mass spectrometer
(Waters, Manchester, U.K.) at 10 μL/min using a Harvard Apparatus
syringe pump. The mass spectrometer was controlled by Masslynx 4.1
software (Waters). It was operated in resolution and positive ion
mode and calibrated using sodium iodide. The sample was analyzed for
1 min with a 1 s MS scan time over the m/z range 50–1200 with 2.0 kV capillary voltage, 40
V cone voltage, and 120 °C cone temperature. Leu-enkephalin peptide
(1 ng/μL, Waters) was infused at 10 μL/min as a lock mass
(m/z 556.2766) and measured every
10 s. Spectra were generated in Masslynx 4.1 by combining multiple
scans, and peaks were centered using automatic peak detection with
lock mass correction.
5
Chemical Profiling of Plant Extracts
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The chemical composition of the extract was determined using the Acquity UPLC system (Waters Corporation, Milford, MA, USA) coupled with a Quadrupole/Time of Flight system (QTOF). Chromatographic runs were performed on a Waters Acquity UPLC BEH column (150 × 2.1 mm, 1.7 µm) injected with 5 µL of the extract solution, with the temperature adjusted to 40 °C. The binary gradient elution system consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), with a linear gradient from 2% to 95% of B (0–15) min) and a flow rate of 0.3 mL/min [80 (link)].
The ESImode was acquired in the range of 110–1180 Da, with the source temperature fixed at 120 °C, a desolvation temperature of 350 °C, a desolvation gas flow of 500 L/h, an extraction cone of 0.5 V, and a capillary voltage of 2.6 kV; the acquisition mode was MSE, with the instrument controlled by the Masslynx 4.1 software (Waters Corporation, Milford, MA, USA). The precise molecular formula and mass assignments were obtained with Masslynx 4.1 software (Waters Corporation, Milford, MA, USA). The data were compared with those described in the literature at the level of the botanical family of the species. Peak identification was determined by m/z values.
The ESImode was acquired in the range of 110–1180 Da, with the source temperature fixed at 120 °C, a desolvation temperature of 350 °C, a desolvation gas flow of 500 L/h, an extraction cone of 0.5 V, and a capillary voltage of 2.6 kV; the acquisition mode was MSE, with the instrument controlled by the Masslynx 4.1 software (Waters Corporation, Milford, MA, USA). The precise molecular formula and mass assignments were obtained with Masslynx 4.1 software (Waters Corporation, Milford, MA, USA). The data were compared with those described in the literature at the level of the botanical family of the species. Peak identification was determined by m/z values.
6
Metabolomic Profiling with XCMS and MarVis
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The data files raw acquired with the Masslynx 4.1 software (Masslynx 4.1, Waters) were transformed into .cdf files with Databridge tool. Chromatographic data files were processed using the software R (http://www.r-project.org/ ). The XCMS algorithm (www.bioconductor.org; Smith et al., 2006) (link) was used to obtain the peak peaking, grouping, and signal corrections. Metabolite amounts were analysed based on the normalized peak area units relative to the dry weight. To test the metabolomic differences between treatments, a nonparametric Kruskal-Wallis test (p < 0.01) was done. Partial least square discriminant analysis and heat map analysis were performed with the metaboAnalyst 4.0 (Chong et al., 2018) (link). Adduct and isotope correction, filtering, clustering, exact mass mapping and metabolic pathway exploration was carried out with the packages MarVis filter, MarVis cluster and MarVis pathway that are integrated in the Marvis suit 2.0 (Kaever et al., 2014) . Metabolite identification was carried out based on exact mass accuracy and fragmentation spectra matching with different online database. The database kegg (https://www.genome.jp/kegg/) was used for exact mass identity and for fragmentation spectrum analysis, the Massbank and the Metlin databases were used (www.massbank.jp; www.masspec.scripps.edu).
7
Native Mass Spectrometry of Protein Complexes
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Native MS experiments were performed on a Waters Micromass Q-ToF Ultima mass spectrometer modified for transmission of high masses84 (link). For this, the storage buffer of 20 µL protein solution was exchanged to 200 mM ammonium acetate using Micro Bio-Spin 6 gel filtration columns (BioRad) or Vivaspin 500 filtration units with a molecular weight cut-off of 10 kDa (Sartorius AG). The proteins were then diluted to concentrations of 5-30 µM. 4 µL of the protein (complexes) were loaded into gold-coated glass capillaries prepared in-house76 (link) and directly introduced into the mass spectrometer. The following parameters were used for data acquisition: capillary voltage, 1.3 – 1.7 kV; sample cone voltage, 80 V; RF lense voltage, 80 V; collision voltage, 10 – 50 V. Mass spectra were processed using MassLynx 4.1 (Waters), externally calibrated using caesium iodide solution (100 mg/mL) and analysed using MassLynx 4.1, Massign software (version 11/14/2014)85 (link) and an in-house written deconvolution macro. All theoretically calculated and experimentally determined masses are given in Supplementary Table 2 .
8
Metabolomic Profiling of Treated Samples
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Data in the.raw format acquired with the Masslynx 4.1 software (Masslynx 4.1, Waters) were transformed into.cdf files using the Databridge tool. Chromatographic signals were processed using the software R for statistical purposes (http://www.r-project.org/ ). Signals from positive and negative electrospray ionization (ESI+; ESI−) analysis were processed separately. Peak peaking, grouping and signal corrections were developed by applying the XCMS algorithm (www.bioconductor.org ; Smith et al., 2006 (link)). Metabolite amounts were analyzed based on the normalized peak area units relative to the dry weight. A non-parametric Kruskal-Wallis test (p < 0.05) was performed to test the metabolomic differences between treatments. Significance was assessed by the absence of overlapping in the box-and-plot graphical method. Principal component analysis and heat map analysis were carried out with the Mar-Vis Suit 2.0 software (Kaever et al., 2015 (link)), a tool for clustering and visualizing metabolic biomarkers. Adduct, isotope correction, clustering and color heat map visualization was also performed by using the packages MarVis Filter and MarVis Cluster. Data were combined for the PCA and heat map analysis but processed separately for compound identification and quantification.
9
Metabolomic Analysis of Sample Treatments
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Data were acquired in centroid mode and subsequently transformed into cdf files using the Databridge from MassLynx 4.1 software (MassLynx 4.1, Waters). Chromatographic signals were processed using the software R for statistical purposes2 . Signals from ESI+ and ESI- were processed separately. Peak peaking, grouping and signal corrections were performed using the XCMS algorithm (Smith et al., 2006 (link)). Metabolite amounts were analyzed on the basis of normalized peak area units relative to the dry weight. The Kruskal–Wallis test (p < 0.05) was applied to analyze the metabolomic differences between treatments. To determine a global behavior of the signals, principal component analyses (PCA) plots were generated using the Multibase 2015 algorithm3 . Statistical and heat map analysis were performed using the MarVis Suit 2.0 software tool for clustering and visualization of metabolic biomarkers (Kaever et al., 2014 (link)). Adduct, isotope correction, clustering, and color heat map visualization were also performed by using associated software packages MarVis Filter and MarVis Cluster.
10
Untargeted Metabolomics Workflow
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Data files from the LC-MS analysis were converted into the NetCDF file format using the Databridge software from MassLynx 4.1 (Waters). The XCMS software was used to perform peak detection and alignment of the LC-MS chromatograms, in R studio. 34, 35 Data processing was performed by excluding all the m/z value with less than 279.1451 (the m/z value corresponding to the monoprotonated probe with no captured metabolite). Features more abundant in the control sample and less than five-fold increase in the feces sample set were eliminated from the data analysis. From each feature 279.1451 Da were subtracted (corresponding to the mass of the probe) and compared to the human metabolome database in order to find plausible candidates for the parent metabolites.
Metabolite validation was performed using MassLynx 4.1 (Waters). Based on the mass and retention time from metabolite-conjugates library, the extracted ion chromatogram (EIC) for each metabolite conjugate was used to confirm the identity of the corresponding metabolite and the correct regioisomers.
Metabolite validation was performed using MassLynx 4.1 (Waters). Based on the mass and retention time from metabolite-conjugates library, the extracted ion chromatogram (EIC) for each metabolite conjugate was used to confirm the identity of the corresponding metabolite and the correct regioisomers.
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