4 Notes
1. Be sure to calculate how many plants you will need to grow for
the number of samples you plan to perform. To obtain 100 mg
of the freeze-dried guard cell preparation for one sample, we
needed approximately 300 fully expanded leaves from
100 plants. We performed extraction from combined freezedried guard cell samples from 100 plants at 72 h during SAR
response, and we collected four biological replicates. For metabolomics, in general it is recommended to collect four to six
biological replicates.
2. To obtain guard cell samples, we used a technique developed in
our lab [7]. It is important to remove the mid-vein of the leaves
before blending in order to remove the large xylem cells of the
vasculature tissue. Leaf mid-veins can be removed using a razor
blade or scalpel. The pore size of the 200 μm nylon mesh allows
for mesophyll and vascular cells to pass through, while retaining the guard cells from the epidermal fragments. Guard cell
viability was verified by staining with fluorescein diacetate
(FDA), which showed that guard cells remained intact and
viable. Only viable cells can hydrolyze the non-fluorescent
FDA to fluorescent fluorescein. Dead cells can neither accumulate nor hydrolyze FDA.
3. We developed a protocol to simultaneously extract metabolites,
lipids, and proteins from a single whole leaf or guard cell
sample. In this procedure, a chloroform and methanol mixture
is added to samples that are in an aqueous methanol solution.
This process induces the formation of two solvent layers: an
upper aqueous phase containing the hydrophilic metabolites,
and a lower organic phase which contains the lipids and other
hydrophobic metabolites [10]. The lower organic phase can be
blown dry by nitrogen gas, and solubilize in isopropanol for
lipidomic analyses [11]. In the middle between the two solvent
layers, there are proteins that can be used for Western blot or
MS analyses. Here we drew off the upper aqueous phase and
immediately dried it using a speed vac to obtain the metabolites
for this analysis.
4. To enhance depth and coverage of metabolome, we used the
Acquire X MS
n data acquisition technology. This allowed for
identification of a greater number of low abundance metabolites by using an iterative fragmentation process targeted by a
sequentially updated inclusion list, as shown in Fig. 2. For
metabolites, MS
2 is often used. For lipids, MS
3 is usually used
in the method. Before collecting Acquire X MS
n data, purge
solution and needle wash to remove air. Ensure there are no
bubbles at the bottom of any injection vials. Before using the
Untargeted Metabolomics of Arabidopsis Stomatal Immunity
421
1. Be sure to calculate how many plants you will need to grow for
the number of samples you plan to perform. To obtain 100 mg
of the freeze-dried guard cell preparation for one sample, we
needed approximately 300 fully expanded leaves from
100 plants. We performed extraction from combined freezedried guard cell samples from 100 plants at 72 h during SAR
response, and we collected four biological replicates. For metabolomics, in general it is recommended to collect four to six
biological replicates.
2. To obtain guard cell samples, we used a technique developed in
our lab [7]. It is important to remove the mid-vein of the leaves
before blending in order to remove the large xylem cells of the
vasculature tissue. Leaf mid-veins can be removed using a razor
blade or scalpel. The pore size of the 200 μm nylon mesh allows
for mesophyll and vascular cells to pass through, while retaining the guard cells from the epidermal fragments. Guard cell
viability was verified by staining with fluorescein diacetate
(FDA), which showed that guard cells remained intact and
viable. Only viable cells can hydrolyze the non-fluorescent
FDA to fluorescent fluorescein. Dead cells can neither accumulate nor hydrolyze FDA.
3. We developed a protocol to simultaneously extract metabolites,
lipids, and proteins from a single whole leaf or guard cell
sample. In this procedure, a chloroform and methanol mixture
is added to samples that are in an aqueous methanol solution.
This process induces the formation of two solvent layers: an
upper aqueous phase containing the hydrophilic metabolites,
and a lower organic phase which contains the lipids and other
hydrophobic metabolites [10]. The lower organic phase can be
blown dry by nitrogen gas, and solubilize in isopropanol for
lipidomic analyses [11]. In the middle between the two solvent
layers, there are proteins that can be used for Western blot or
MS analyses. Here we drew off the upper aqueous phase and
immediately dried it using a speed vac to obtain the metabolites
for this analysis.
4. To enhance depth and coverage of metabolome, we used the
Acquire X MS
n data acquisition technology. This allowed for
identification of a greater number of low abundance metabolites by using an iterative fragmentation process targeted by a
sequentially updated inclusion list, as shown in Fig. 2. For
metabolites, MS
2 is often used. For lipids, MS
3 is usually used
in the method. Before collecting Acquire X MS
n data, purge
solution and needle wash to remove air. Ensure there are no
bubbles at the bottom of any injection vials. Before using the
Untargeted Metabolomics of Arabidopsis Stomatal Immunity
421