Data generation and analysis with SIRIUS 4 on two biological case studies
The procedures for the metabolomics analysis performed in the present work has been described previously for skin[1] and fecal material[2]. The detailed step by step instructions to reproduce the biological case studies presented in this work are given below.
This protocol summarizes the workflow on how to get from sample to the results described in the paper Sirius 4
Ethanol of LC-MS (Optima) grade (ThermoFisher Scientic) for swabbing and sample extraction
CAUTION: ethanol is highly ammable and irritating to eyes and skin.
Water of LC-MS (Optima) grade (ThermoFisher Scientic).
ESI-L Low Concentration Tuning Mix (Agilent Technologies, CA, USA) for external calibration
of the MaXis II QTOF mass spectrometer
Hexakis(1H,1H,3H-tetrauoropropoxy)phosphazene (Synquest Laboratories, FL, USA), m/z
922.009798, for internal calibration (lockmass) of the MaXis II QTOF mass spectrometer.
CAUTION: This compound is irritating to eyes and skin.
Acetonitrile LC-MS (Optima) grade (ThermoFisher Scientic). CAUTION: Acetonitrile is highly
ammable.
Formic acid of LC-MS grade, Optima grade (ThermoFisher Scientic). CAUTION: Formic acid
is highly corrosive.
(Skin sampling): Cotton swabs, model Puritan® (Spectrum, CA, USA), reference 150-533353
or 150-2971696.
(Faeces sampling): BBL culture swabs (Becton, Dickinson and Company, Sparks, MD).
Deep-wells microtiter plate, 2 mL, polypropylene, model NUNC (ThermoFisher Scientic, MA,
USA) reference 278753 or an equivalent generic 96 wells microtiter plate, 400 µL, polystyrene.
Reusable lid for a 96 wells plate (ThermoFisher Scientic), reference 351190 or equivalent.
Stainless steel tweezers.
Single or multi channel 20-200 µL micropipette.
Plate mat for storage, model Corning Storage Mat III (ThermoFisher Scientic, MA, USA).
Benchtop vacuum concentrator compatible with 96-well plates evaporation (Centrivap Labconco,
MO, USA).
Reverse phase C18 LC column 1.7 µm particle size, 50 2.1 mm (Phenomenex, CA, USA) or
equivalent.
Ultra high-performance liquid chromatography (UHPLC) system coupled to a tandem mass
spectrometer with an ESI source; for the biological studies described here we have used UltiMate
3000 UHPLC (Dionex, Idstein, Germany) coupled to MaXis II QTOF (Bruker Daltonics,
Bremen, Germany).
Sampling of skin
as possible across spots.
CRITICAL STEP: During the placing of the swab and the cutting of the cotton bud, use
aluminium fold lm to cover the rest of the plate to prevent cross-contamination of other
wells.
reagents, and ideally by the same operator.
Sampling of stool
The sampling is carried out by individuals as described in [2]. Samples are
collected using swabs and returned by mail. Samples collected outside of the United States are
shipped using domestic post within each country to an aggregation site and stored at -80 at the
aggregation site until shipment to the United States. Shipment to the United States is done on dry
ice using a certied shipping service.
Metabolite extraction of skin samples
temperature and longer extraction time. However, this can lead to the degradation of some
molecules.
this step prevent contamination of other wells by covering them with aluminium lm.
that the rotor is properly equilibrated before spinning.
contamination of the plate by covering it with aluminium foil, and avoid undissolved material
upon sample transfer.
any precipitated material.
Metabolite extraction of stool samples
avoid cross contamination of the plate by covering it with aluminium foil, and avoid undissolved
material upon sample transfer.
swabs and extraction solvent onto each plate.
before spinning.
ensure that the rotor is properly equilibrated before spinning.
Mass spectrometry analysis
5 µL for fecal samples). Prime the injection system multiple time.
guideline. For the MaXis II QTOF, add the solution containing internal lockmass (m/z 922.009)
in the ESI source in the positive ion mode, following the the manufacturer's guideline.
V; ion source temperature, 180 ; dry gas ow, 9 L/min; spectra rate acquisition, 3 spectra/s.
Perform MS/MS fragmentation of the seven most intense selected ions per spectrum (TOP7)
using ramped collision-induced dissociation (CID) energy, ranging from 16 to 48 eV, to get
diverse fragmentation patterns. Set MS/MS exclusion after 3 spectra to be released after 30 s.
Set an MS/MS exclusion list for the mass range of m/z 921.5924.5 to exclude the lockmass, and
if needed include other adducts of the lockmass if their intensities are in the range of MS/MS
threshold.
thresholds appropriate for the instrument (e.g. below few thousand counts for the MaXis II
QTOF instrument). If high abundance contaminants are present, consider additional source
cleaning, replacement of mobile phases, glassware, LC column or tubing, as needed.
samples (coefficient of variation, CV, below 0.1-0.3). Check for carryover by inspecting the blank
that followed a QC sample. If needed, adjust the injection volume to prevent chromatographic
saturation (peak broadening), and mass spectrometer detector saturation (peak flattening). If
needed, optimize the LC gradient to ensure appropriate separation of representative features,
and the number of needle/injector washes to reduce carry-over below 1%.
samples order should be randomized.
not eluted completely by the mobile phase. Typically, this clean-up run is composed of multiple
mobile phase composition variation according to the manufacturer's guidelines.
(ii) that the calibration is stable (< 10 ppm for MaXis II QTOF). If needed, stop the LC-MS
sequence, and calibrate the instrument according to manufacturer guidelines. For the MaXis II
QTOF, lock mass calibrant must be added every 12 hours. If the LC-MS sequence was stopped,
run a QC sample and a blank prior continuing the rest of your analysis.
Overlap features across samples, such as the internal standard, and assess the stability of the
chromatography (retention shift in seconds), and the MS calibration (m/z window in ppm).
in centroid mode (select Filters: Peak Picking, MS-Levels 1-2).
Data analysis
Creating feature tables. Multiple software packages for MS feature extraction exist and can be used;
the present protocol is given for the use of the open-source OpenMS 2.0[3] software utilized for feature
detection in the fecal sampling biological study example and MZmine2[4] used in the skin sampling
biological study. The recommended settings were found to be appropriate for the data obtained in
our experiments; however, experienced users may nd changes in parameters necessary, especially
if dierent instrumentation is used.
Creating feature tables with OpenMS.
chromatographic peak full width at half-maximum (FWHM) value is set at 20, and the mass
error is set at 30 ppm.
based on specics of each experiment.
Creating feature tables with MZmine2.
Mass detection
based on specics of each experiment.
Molecular networking
discarded.
the nodes appeared in each other's respective top 10 most similar nodes.
level 2/3 according to the proposed minimum standards in metabolomics[7].
the status page.
as described in reference [6] by assessing dierences in parent mass and fragmentation patterns.
(RTs) and MS/MS spectra need to be compared to those experimentally obtained for the
putatively annotated compounds.
Determining the molecular structure with SIRIUS 4
node.
MS/MS peaks will belong to the consensus spectrum obtained from clustering multiple
experimental spectra and thus may dier from MS/MS patterns in individual files.
Caution must be used to ensure that the appropriate spectrum is used for further analysis.
would result in longer computing time)
Under the "Compounds" tab (left hand side), the currently active job is selected.
displayed below for each formula or could be viewed individually in the corresponding tabs.
experimental spectrum is not present in selected source databases.
not be the case and researcher's discretion and expertise are essential for selecting the potential
candidate molecules for further consideration and interpretation.
The sampling time for skin swabbing and faeces sampling is generally under one minute per sample.
The total time necessary for the samples preparation and data analysis depends on the number of
samples. Typical timing is approximately one hour for placing swabs into 96-well plate, 6 hours to
12 hours for metabolite extraction, 24 hours for LC-MS analysis, 2 hours for MS feature extraction
(strongly depends on the computational power of the system that runs the software), 6 hours
for preparing and running GNPS job. The time for network-based annotation propagation can vary
widely depending on the analysis circumstances. The time for a single SIRIUS/CSI:FingerID job for
a single spectrum processing is typically few seconds on a personal computer. A detailed breakdown
of timing for specic experimental steps is given in publication of Sirius 4.
The users should follow general good laboratory practices for the analysis to avoid issues such
as contamination. For detailed troubleshooting guidance specic to the LC-MS/MS analysis, the
user should refer to the manuals of the instrumentation that is used in the experiment. Additional
troubleshooting guidelines specic to the described workow are given in [1]. The troubleshooting
tips for the GNPS-based molecular networking and annotation could be accessed at: https://
ccms-ucsd.github.io/GNPSDocumentation/troubleshooting/
The anticipated results should be comparable to those described in the given examples for the two
biological case studies.
Bioinf 11, 395 (2010).
Posted 20 Sep, 2019
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Data generation and analysis with SIRIUS 4 on two biological case studies
The procedures for the metabolomics analysis performed in the present work has been described previously for skin[1] and fecal material[2]. The detailed step by step instructions to reproduce the biological case studies presented in this work are given below.
This protocol summarizes the workflow on how to get from sample to the results described in the paper Sirius 4
Ethanol of LC-MS (Optima) grade (ThermoFisher Scientic) for swabbing and sample extraction
CAUTION: ethanol is highly ammable and irritating to eyes and skin.
Water of LC-MS (Optima) grade (ThermoFisher Scientic).
ESI-L Low Concentration Tuning Mix (Agilent Technologies, CA, USA) for external calibration
of the MaXis II QTOF mass spectrometer
Hexakis(1H,1H,3H-tetrauoropropoxy)phosphazene (Synquest Laboratories, FL, USA), m/z
922.009798, for internal calibration (lockmass) of the MaXis II QTOF mass spectrometer.
CAUTION: This compound is irritating to eyes and skin.
Acetonitrile LC-MS (Optima) grade (ThermoFisher Scientic). CAUTION: Acetonitrile is highly
ammable.
Formic acid of LC-MS grade, Optima grade (ThermoFisher Scientic). CAUTION: Formic acid
is highly corrosive.
(Skin sampling): Cotton swabs, model Puritan® (Spectrum, CA, USA), reference 150-533353
or 150-2971696.
(Faeces sampling): BBL culture swabs (Becton, Dickinson and Company, Sparks, MD).
Deep-wells microtiter plate, 2 mL, polypropylene, model NUNC (ThermoFisher Scientic, MA,
USA) reference 278753 or an equivalent generic 96 wells microtiter plate, 400 µL, polystyrene.
Reusable lid for a 96 wells plate (ThermoFisher Scientic), reference 351190 or equivalent.
Stainless steel tweezers.
Single or multi channel 20-200 µL micropipette.
Plate mat for storage, model Corning Storage Mat III (ThermoFisher Scientic, MA, USA).
Benchtop vacuum concentrator compatible with 96-well plates evaporation (Centrivap Labconco,
MO, USA).
Reverse phase C18 LC column 1.7 µm particle size, 50 2.1 mm (Phenomenex, CA, USA) or
equivalent.
Ultra high-performance liquid chromatography (UHPLC) system coupled to a tandem mass
spectrometer with an ESI source; for the biological studies described here we have used UltiMate
3000 UHPLC (Dionex, Idstein, Germany) coupled to MaXis II QTOF (Bruker Daltonics,
Bremen, Germany).
Sampling of skin
as possible across spots.
CRITICAL STEP: During the placing of the swab and the cutting of the cotton bud, use
aluminium fold lm to cover the rest of the plate to prevent cross-contamination of other
wells.
reagents, and ideally by the same operator.
Sampling of stool
The sampling is carried out by individuals as described in [2]. Samples are
collected using swabs and returned by mail. Samples collected outside of the United States are
shipped using domestic post within each country to an aggregation site and stored at -80 at the
aggregation site until shipment to the United States. Shipment to the United States is done on dry
ice using a certied shipping service.
Metabolite extraction of skin samples
temperature and longer extraction time. However, this can lead to the degradation of some
molecules.
this step prevent contamination of other wells by covering them with aluminium lm.
that the rotor is properly equilibrated before spinning.
contamination of the plate by covering it with aluminium foil, and avoid undissolved material
upon sample transfer.
any precipitated material.
Metabolite extraction of stool samples
avoid cross contamination of the plate by covering it with aluminium foil, and avoid undissolved
material upon sample transfer.
swabs and extraction solvent onto each plate.
before spinning.
ensure that the rotor is properly equilibrated before spinning.
Mass spectrometry analysis
5 µL for fecal samples). Prime the injection system multiple time.
guideline. For the MaXis II QTOF, add the solution containing internal lockmass (m/z 922.009)
in the ESI source in the positive ion mode, following the the manufacturer's guideline.
V; ion source temperature, 180 ; dry gas ow, 9 L/min; spectra rate acquisition, 3 spectra/s.
Perform MS/MS fragmentation of the seven most intense selected ions per spectrum (TOP7)
using ramped collision-induced dissociation (CID) energy, ranging from 16 to 48 eV, to get
diverse fragmentation patterns. Set MS/MS exclusion after 3 spectra to be released after 30 s.
Set an MS/MS exclusion list for the mass range of m/z 921.5924.5 to exclude the lockmass, and
if needed include other adducts of the lockmass if their intensities are in the range of MS/MS
threshold.
thresholds appropriate for the instrument (e.g. below few thousand counts for the MaXis II
QTOF instrument). If high abundance contaminants are present, consider additional source
cleaning, replacement of mobile phases, glassware, LC column or tubing, as needed.
samples (coefficient of variation, CV, below 0.1-0.3). Check for carryover by inspecting the blank
that followed a QC sample. If needed, adjust the injection volume to prevent chromatographic
saturation (peak broadening), and mass spectrometer detector saturation (peak flattening). If
needed, optimize the LC gradient to ensure appropriate separation of representative features,
and the number of needle/injector washes to reduce carry-over below 1%.
samples order should be randomized.
not eluted completely by the mobile phase. Typically, this clean-up run is composed of multiple
mobile phase composition variation according to the manufacturer's guidelines.
(ii) that the calibration is stable (< 10 ppm for MaXis II QTOF). If needed, stop the LC-MS
sequence, and calibrate the instrument according to manufacturer guidelines. For the MaXis II
QTOF, lock mass calibrant must be added every 12 hours. If the LC-MS sequence was stopped,
run a QC sample and a blank prior continuing the rest of your analysis.
Overlap features across samples, such as the internal standard, and assess the stability of the
chromatography (retention shift in seconds), and the MS calibration (m/z window in ppm).
in centroid mode (select Filters: Peak Picking, MS-Levels 1-2).
Data analysis
Creating feature tables. Multiple software packages for MS feature extraction exist and can be used;
the present protocol is given for the use of the open-source OpenMS 2.0[3] software utilized for feature
detection in the fecal sampling biological study example and MZmine2[4] used in the skin sampling
biological study. The recommended settings were found to be appropriate for the data obtained in
our experiments; however, experienced users may nd changes in parameters necessary, especially
if dierent instrumentation is used.
Creating feature tables with OpenMS.
chromatographic peak full width at half-maximum (FWHM) value is set at 20, and the mass
error is set at 30 ppm.
based on specics of each experiment.
Creating feature tables with MZmine2.
Mass detection
based on specics of each experiment.
Molecular networking
discarded.
the nodes appeared in each other's respective top 10 most similar nodes.
level 2/3 according to the proposed minimum standards in metabolomics[7].
the status page.
as described in reference [6] by assessing dierences in parent mass and fragmentation patterns.
(RTs) and MS/MS spectra need to be compared to those experimentally obtained for the
putatively annotated compounds.
Determining the molecular structure with SIRIUS 4
node.
MS/MS peaks will belong to the consensus spectrum obtained from clustering multiple
experimental spectra and thus may dier from MS/MS patterns in individual files.
Caution must be used to ensure that the appropriate spectrum is used for further analysis.
would result in longer computing time)
Under the "Compounds" tab (left hand side), the currently active job is selected.
displayed below for each formula or could be viewed individually in the corresponding tabs.
experimental spectrum is not present in selected source databases.
not be the case and researcher's discretion and expertise are essential for selecting the potential
candidate molecules for further consideration and interpretation.
The sampling time for skin swabbing and faeces sampling is generally under one minute per sample.
The total time necessary for the samples preparation and data analysis depends on the number of
samples. Typical timing is approximately one hour for placing swabs into 96-well plate, 6 hours to
12 hours for metabolite extraction, 24 hours for LC-MS analysis, 2 hours for MS feature extraction
(strongly depends on the computational power of the system that runs the software), 6 hours
for preparing and running GNPS job. The time for network-based annotation propagation can vary
widely depending on the analysis circumstances. The time for a single SIRIUS/CSI:FingerID job for
a single spectrum processing is typically few seconds on a personal computer. A detailed breakdown
of timing for specic experimental steps is given in publication of Sirius 4.
The users should follow general good laboratory practices for the analysis to avoid issues such
as contamination. For detailed troubleshooting guidance specic to the LC-MS/MS analysis, the
user should refer to the manuals of the instrumentation that is used in the experiment. Additional
troubleshooting guidelines specic to the described workow are given in [1]. The troubleshooting
tips for the GNPS-based molecular networking and annotation could be accessed at: https://
ccms-ucsd.github.io/GNPSDocumentation/troubleshooting/
The anticipated results should be comparable to those described in the given examples for the two
biological case studies.
Bioinf 11, 395 (2010).
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