4. Employ the Full MS scan method to acquire data from the
Acquire X blank, the Acquire X pool sample, and each individual metabolite sample for both positive and negative mode.
Method details are as follows: Global Settings: method duration (min) ¼ 10, positive ion spray voltage (V) ¼ 3500, negative ion spray voltage (V) ¼ 2500, sheath gas (arb) ¼ 50, aux
gas (arb) ¼ 10, sweep gas (arb) ¼1, ion transfer tube temperature (
C) ¼ 325, vaporizer temperature (
C) ¼ 350, cycle time
(s) ¼ 0.6. Master Scan: MS
n Level ¼ 1, detector type ¼ Orbitrap, Orbitrap resolution ¼ 120 k, scan range (m/z) ¼ 55–550,
maximum injection time (ms) ¼ 50, AGC target ¼ 200,000,
RF lens (%) ¼ 50, and polarity ¼ positive or negative for
different modes.
5. A positive or negative mode MS
n method is employed in
acquiring MS
2 data to obtain product fragments that are used
for identifying compounds from database matches. The specific
method is as follows: The Global Settings and Master Scan are
identical to the previous Full MS scan method. Filter dynamic
exclusion: exclude after n times ¼ 1, exclusion duration (s) ¼ 3,
mass tolerance ¼ 10 ppm. Filter Targeted inclusion mass list:
MS tolerance 10 ppm. The dynamic exclusion list and the
targeted inclusion list is made from the Full MS scan method.
ddMS
n Scan: MS
n level ¼ 2 (for metabolites), isolation
mode ¼ Quadrupole, isolation window ¼ 0.7, scan range
(m/z) ¼ 55–550, activation type ¼ HCD, collision energy
mode ¼ stepped, collision energies (%) ¼ 27, 30, 33, detector
type ¼ Orbitrap, Orbitrap resolution ¼ 7.5 K, maximum injection time (ms) ¼ 20, AGC target ¼ 50,000, polarity ¼ positive
or negative for different modes.
3.5 Data Analysis
(See Note 5)
1. We used Compound Discover™ 3.0 Software from Thermo
Fisher Scientific for untargeted metabolomics data analyses.
The first step will be Starting a New Study using the Analysis
Wizard. You will be prompted to set up a folder for your study
and the analysis will be saved to that folder.
2. The next step in Starting a New Study will be to create a
workflow. You can choose from a list of default workflows,
but we found it was better to create our own workflow for
untargeted metabolomics. The goal of any Untargeted Metabolomics workflow is to find and identify the differences
between samples. The user can design the workflow to be
appropriate for their study. When designing a workflow, you
will need to choose nodes from ten areas. These ten areas
include (1) input/output, (2) spectrum processing, (3) trace
creation, (4) compound detection, (5) peak area refinement,
(6) compound identification, (7) pathway mapping, (8) compound scoring, (9) expected compounds, and (10) postprocessing. We chose the following nodes from these areas:
Untargeted Metabolomics of Arabidopsis Stomatal Immunity
419
Acquire X blank, the Acquire X pool sample, and each individual metabolite sample for both positive and negative mode.
Method details are as follows: Global Settings: method duration (min) ¼ 10, positive ion spray voltage (V) ¼ 3500, negative ion spray voltage (V) ¼ 2500, sheath gas (arb) ¼ 50, aux
gas (arb) ¼ 10, sweep gas (arb) ¼1, ion transfer tube temperature (
C) ¼ 325, vaporizer temperature (
C) ¼ 350, cycle time
(s) ¼ 0.6. Master Scan: MS
n Level ¼ 1, detector type ¼ Orbitrap, Orbitrap resolution ¼ 120 k, scan range (m/z) ¼ 55–550,
maximum injection time (ms) ¼ 50, AGC target ¼ 200,000,
RF lens (%) ¼ 50, and polarity ¼ positive or negative for
different modes.
5. A positive or negative mode MS
n method is employed in
acquiring MS
2 data to obtain product fragments that are used
for identifying compounds from database matches. The specific
method is as follows: The Global Settings and Master Scan are
identical to the previous Full MS scan method. Filter dynamic
exclusion: exclude after n times ¼ 1, exclusion duration (s) ¼ 3,
mass tolerance ¼ 10 ppm. Filter Targeted inclusion mass list:
MS tolerance 10 ppm. The dynamic exclusion list and the
targeted inclusion list is made from the Full MS scan method.
ddMS
n Scan: MS
n level ¼ 2 (for metabolites), isolation
mode ¼ Quadrupole, isolation window ¼ 0.7, scan range
(m/z) ¼ 55–550, activation type ¼ HCD, collision energy
mode ¼ stepped, collision energies (%) ¼ 27, 30, 33, detector
type ¼ Orbitrap, Orbitrap resolution ¼ 7.5 K, maximum injection time (ms) ¼ 20, AGC target ¼ 50,000, polarity ¼ positive
or negative for different modes.
3.5 Data Analysis
(See Note 5)
1. We used Compound Discover™ 3.0 Software from Thermo
Fisher Scientific for untargeted metabolomics data analyses.
The first step will be Starting a New Study using the Analysis
Wizard. You will be prompted to set up a folder for your study
and the analysis will be saved to that folder.
2. The next step in Starting a New Study will be to create a
workflow. You can choose from a list of default workflows,
but we found it was better to create our own workflow for
untargeted metabolomics. The goal of any Untargeted Metabolomics workflow is to find and identify the differences
between samples. The user can design the workflow to be
appropriate for their study. When designing a workflow, you
will need to choose nodes from ten areas. These ten areas
include (1) input/output, (2) spectrum processing, (3) trace
creation, (4) compound detection, (5) peak area refinement,
(6) compound identification, (7) pathway mapping, (8) compound scoring, (9) expected compounds, and (10) postprocessing. We chose the following nodes from these areas:
Untargeted Metabolomics of Arabidopsis Stomatal Immunity
419