Samples were analyzed by a hybrid quadrupole time-of-flight mass spectrometer (maXis Impact, Bruker Daltonics, Billerica, MA, USA) equipped with an electrospray ionization (ESI) source (drying gas temperature (nitrogen)—180 °C; drying gas flow rate—4 L/min; capillary voltage—4000 V; focusing voltage—500 V; electrospray pressure—0.3 bar; low-pressure funnel RF voltage—90V; high-pressure funnel RF voltage—120 V). The mass spectrometer was set up to prioritize the detection of ions with a mass-to-charge ratio (m/z) ranging from 45 to 900, with a mass accuracy of 1–3 parts per million (ppm). The spectra were recorded in the positive ion charge detection mode. The samples were injected into the ESI source using a glass syringe (Hamilton Bonaduz AG, Bonaduz, Switzerland) connected to a syringe injection pump (KD Scientific, Holliston, MA, USA). The rate of sample flow to the ionization source was 180 µL/h. Mass spectra were obtained using DataAnalysis version 4.1 (Bruker Daltonics) to summarize one-minute signals.
Dataanalysis version 4
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DataAnalysis version 4.1 is a software application for data analysis and visualization. It provides tools for processing, analyzing, and presenting data from various scientific instruments and experiments.
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Lab products found in correlation
Maxis impact, by Bruker (1 mentions)
Syringe injection pump, by KD Scientific (1 mentions)
Matlab version r2010a, by MathWorks (1 mentions)
Flexanalysis, by Bruker (1 mentions)
Prism 6, by GraphPad (1 mentions)
Trap control, by Bruker (1 mentions)
Microtof mass spectrometer, by Bruker (1 mentions)
Ultimate 3000 series, by Thermo Fisher Scientific (1 mentions)
Amazon speed, by Bruker (1 mentions)
Acquity uplc beh c18 column, by Waters Corporation (1 mentions)
Rezex roa column, by Phenomenex (1 mentions)
1100 series hplc instrument, by Hewlett-Packard (1 mentions)
Esquire lc ms ion trap mass spectrometer, by Bruker (1 mentions)
Spss statistics 23, by IBM (1 mentions)
Nanoacquity ultra performance liquid chromatography system, by Waters Corporation (1 mentions)
Amazon speed ion trap mass spectrometry, by Bruker (1 mentions)
Amazon etd ion trap, by Bruker (1 mentions)
Peaks software, by Bioinformatics Solutions (1 mentions)
Solarix xr 9.4t, by Bruker (1 mentions)
Flexcontrol software version 3, by Bruker (1 mentions)
17 protocols using dataanalysis version 4
1
Hybrid Quadrupole Time-of-Flight Mass Spectrometry
2
Mass Spectrometry Data Preprocessing
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Mass spectra were obtained by summarizing one-minute signals using DataAnalysis version 4.1 (Bruker Daltonics). Mass spectra preprocessing including recalibration, peak detection, and peak intensity calculation were carried out by DataAnalysis software using the following parameters: peak width, 2; signal-to-noise ratio, 2; relative and absolute threshold intensity, 0.01% and 100, respectively. The resulting mass spectrometry peak masses were pooled and processed using Matlab version R2010a (MathWorks, Natick, MA, USA). The alignment of mass spectrometry data was performed as described previously [25 (link)]. If mass difference was not exceeding 0.01 Da the peaks were considered as related to the same metabolite ion. Peaks that were below the detection limit in <80% of samples in each group were removed from the analysis. Mass peak intensities were normalized as described previously [24 (link)].
3
Comprehensive Mass Spectrometry Data Analysis
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DataAnalysis version 4.1 (Bruker Daltonics, Bremen, Germany) was used for manual inspection and the processing of the HPLC–TOFMS mass spectra and chromatograms, internal recalibration of the spectra, and picking accurate masses. MSConvert Version: 3.0.19 (a tool of ProteoWizard; Palo Alto, CA, USA) [28 (link)] was used to convert the files to .mzxml format, using the parameter “vendor” of the peak picking filter. The IROA kit comes with dedicated software, ClusterFinder™ V3 (IROA Technologies LLC®, Bolton, MA, USA). It was used to establish a library based on the measurements of the LTRS runs. The untargeted analysis was done using MZmine 2 [29 (link)], where the chromatographic peaks were detected, automatically integrated, deconvoluted, aligned, and filtered. The integrated peak areas were extracted for further statistical evaluation.
4
Conotoxin Sequencing via De Novo MS/MS
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Manual de novo sequencing strategy was followed to sequence the conotoxins from the raw data obtained from LC-MS-MS. Data were analysed in Data Analysis version 4.1 and Flex analysis (Bruker Daltonics). The daughter ions generated from singly and doubly charged parent ions were carefully analysed to derive the amino acid sequences of the venom complex (Rajesh, 2015 (link), Rajesh et al., 2019 (link)).
5
NMR, MALDI-TOF, and ESI-IT MS Data Analysis
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NMR spectra were processed using 1D WINNMR, version 6.2 (Bruker Analytische Messtechnik GmbH, Rheinstetten, Germany). MALDI-TOF MS data were acquired and processed by “Flex Control” and “Flex Analysis” version 3.0. For data acquisition and subsequent analysis of ESI-IT MS data, the software “Trap Control” and “Data Analysis” version 4.1 (all Bruker Daltonics GmbH) were used, respectively.
Statistical analyses were performed using GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). Mean values and standard deviations were calculated. For the verification of significances nonparametric and two-tailed t-tests were performed, and significance was indicated by p < 0.05.
Statistical analyses were performed using GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). Mean values and standard deviations were calculated. For the verification of significances nonparametric and two-tailed t-tests were performed, and significance was indicated by p < 0.05.
6
CE-ESI-MS Analysis of Cell Extracts
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Each cell extract was dried
and resuspended in 1 μL of 1% formic acid in liquid chromatography–MS
grade water. CE–ESI-MS was performed as reported previously
using a micrOTOF mass spectrometer (Bruker Daltonics).27 (link) Analyses were conducted in positive ion mode
using a 70.7 cm long CE fused-silica capillary (Polymicro Technologies),
a separation potential of 17 kV, and a sample injection volume of
∼15 nL. Extracted ion electropherograms were exported using
custom scripts in Bruker DataAnalysis version 4.4. Compounds were
identified from the electropherograms by matching the migration order
and mass-to-charge (m/z) values
with standards. In MATLAB, each extracted ion electropherogram was
baseline-subtracted and smoothed with a seven point moving average
filter. Analyte migration times were aligned to corresponding analyte
migration times in a reference mass electropherogram (α1), as
shown inFigure S1 . The alignment used
a linear regression between migration times of a set of amino acids
found in each sample (i.e., glycine, alanine, threonine, leucine/isoleucine,
histidine, phenylalanine). To confirm the presence of dopamine, a
standard mix of 10 μM glycine, alanine, threonine, leucine,
histidine, and phenylalanine in 1% formic acid in water was analyzed
with a 68 cm long CE capillary at 10 kV with and without the addition
of 10 μM dopamine.
and resuspended in 1 μL of 1% formic acid in liquid chromatography–MS
grade water. CE–ESI-MS was performed as reported previously
using a micrOTOF mass spectrometer (Bruker Daltonics).27 (link) Analyses were conducted in positive ion mode
using a 70.7 cm long CE fused-silica capillary (Polymicro Technologies),
a separation potential of 17 kV, and a sample injection volume of
∼15 nL. Extracted ion electropherograms were exported using
custom scripts in Bruker DataAnalysis version 4.4. Compounds were
identified from the electropherograms by matching the migration order
and mass-to-charge (m/z) values
with standards. In MATLAB, each extracted ion electropherogram was
baseline-subtracted and smoothed with a seven point moving average
filter. Analyte migration times were aligned to corresponding analyte
migration times in a reference mass electropherogram (α1), as
shown in
a linear regression between migration times of a set of amino acids
found in each sample (i.e., glycine, alanine, threonine, leucine/isoleucine,
histidine, phenylalanine). To confirm the presence of dopamine, a
standard mix of 10 μM glycine, alanine, threonine, leucine,
histidine, and phenylalanine in 1% formic acid in water was analyzed
with a 68 cm long CE capillary at 10 kV with and without the addition
of 10 μM dopamine.
7
HPLC-MS Analysis of Heimiomyces Extracts
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The extracts obtained from liquid cultures of Heimiomyces sp. were dissolved in acetone to yield a concentration of 10 mg/mL. Analysis of the samples was performed with an analytical HPLC device (Dionex UltiMate 3000 series, Sunnyvale, CA, USA) coupled to an ion trap mass spectrometer (amazon speed™ by Bruker). As mobile phase HPLC grade water and MeCN, both containing 0.1% of formic acid, were used. After injection of 2 μL of the samples, the separation was carried out over an ACQUITY-UPLC® BEH C18 column (50 × 2.1 mm; particle size: 1.7 μm) (Waters) with a flow rate of 600 μL/min. The gradient started at 5% of MeCN, then increased to 100% MeCN in 20 min and remained for 5 min at 100%. To evaluate the obtained chromatograms, the appropriate analysis software (Data Analysis, version 4.4 by Bruker) was used.
8
Purification and Characterization of Peroxidase-Inhibiting Compound
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HPLC was used to purify the compound(s) that inhibits peroxidases and generates H2O2 when added into 1 μM Cu2+. DKG preparations (∼46 mM, pH ∼1 and ∼6) were filtered (0.4 μm, Chromacol), and 40 μl was fractionated on a Phenomenex Rezex ROA column, run (0.5 ml min−1) at 35 °C, routinely with 47 mM H2SO4 as mobile phase. In some experiments, 13 mM TFA [0.1% (v/v)] was used when a volatile mobile phase was required. Degradation products were detected by UV absorbance at various wavelengths.
The major peak of cmpd (1 ) was collected and stored frozen prior to analysis. Mass spectrometry measurements were performed by electrospray on a 12T SolariX Fourier transform mass spectrometer (Bruker Daltonics) equipped with an infinity cell and operating in positive mode. Spectra were the sum of 20 mass analyses and collected with a data size of 4 Mword. Agilent tune mix was used for external calibration. Analysis was achieved with Data Analysis version 4.4 (Bruker Daltonics).
The major peak of cmpd (
9
Robust CE-MS Data Preprocessing
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After mass calibration using the measurement of sodium formate salts at the start of each run, the raw MS-data were converted into NetCDF format (http://www.unidata.ucar.edu/software/netcdf/ ) through the Bruker software (DataAnalysis version 4.0). The NetCDF files were filtered by excluding spectra corresponding to a migration time less than 520 or greater than 3650 seconds prior to preprocessing using the Bioconductor package xcms34 (link) as previously described25 (link). All the standard xcms pipeline parameters were kept to their defaults apart from steps which was set to 3 and bw which was set to 20. In addition, the total number of migration time alignment iterations was set to 5, using the LOESS approach of xcms. The resulting molecule features derived from the execution of the xcms pipeline (in terms of m/z and migration time pairs) were further filtered for their presence across samples by including only those molecule features present in at least 50% of the total samples. The latter ensured the robustness of the initial set of molecule intensities which would be later interrogated for the presence of stable (in terms of intensity) molecule features that would serve as a set of CE-MS internal normalization standards.
10
HPLC-MS Analysis of Anthocyanins and Flavonols
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An analytical Hewlett Packard 1100 series HPLC instrument (Palo Alto, CA, USA) equipped with an autosampler, binary HPLC pump, and UV/Vis detector was used. For HPLC/MS analysis, the HPLC apparatus was interfaced to a Bruker model Esquire-LC/MS ion trap mass spectrometer (Billerica, MA, USA). Mass spectral data were collected with the Bruker software (Bruker Co., DataAnalysis version 4.0, Billerica, MA, USA), which also controlled the instrument and collected the signal at 520 nm. Typical conditions for mass spectral analysis conducted in positive-ion electrospray mode for anthocyanins and negative-ion electrospray mode for flavonols included a capillary voltage of 4000 V, a nebulizing pressure of 30.0 psi, a drying gas flow of 9.0 mL/min, and a temperature of 300 °C. Data were collected in full scan mode over a mass range of m/z 50−1000 at 1.0 s per cycle. Characteristic ions (m/z) were used for peak assignment. For compounds where chemical standards were commercially available, retention times were also used to confirm the identification of components.
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