methanol (LC-MS grade) containing the internal standard
(e.g., 0.5 μg/mL formononetin) to the glass vial.
2. Transfer the glass vial into a shaker with controlled temperature. For heat-stable compounds perform the incubation
through continuous agitation for 30 min at 30–37
C. For
heat-labile compounds perform the incubation at 20–25
C.
3. To remove microsome lipids, transfer 800 μL of eluted samples
in a safe-lock microcentrifuge tube and add 400 μL of chloroform. Vortex for 30 s and shake in a homogenizer, for example
use a Mixer Mill MM 400 (Retsch) or similar for 5 min at
20 Hz frequency. Add 200 μL ultrapure water to separate
phases, vortex and centrifuge at 20,000 Â g for 20 min.
4. Recover 800 μL of the upper phase and transfer into a
new tube.
5. Dry the sample in a vacuum concentrator and resuspend it in
80 μL of 50% (v/v) methanol; centrifuge at 20,000 Â g for
10 min to remove precipitates.
6. Analyze an aliquot of the supernatant by LC-PDA-HRMS (see
Note 8).
4 Notes
1. Do not autoclave solutions described in Subheadings 2.3 and
2.4; store at 4
C for few weeks.
2. Select an adequate yeast strain to perform the transport assays.
For example, S. cerevisiae contains five vacuolar localized
ABCC members (Ycf1p, Bpt1p, Ybt1p/Bat1p, Nft1p, and
Vmr1p) [22]. So, in case of studying a plant ABCC transporter
it is advisable to test yeast strains mutated in one or multiple
ABCCs present in S. cerevisiae and perform transport assays
using microsomes purified from yeasts that heterologous
express the transporter of interest.
3. A critical point to obtain intact and functional yeast microsomes is the yeast growth. Using our experimental conditions,
we obtained good results when OD600 is 3–3.5 after O/N
growth. Furthermore it is suggested to test the physiological
intactness of the isolated microsomes before transport assays.
This can be performed by the vesicles lumen acidification
method as described in [23].
4. Choose as Internal standard (IS) a compound that has similar
chemical properties to your analytes, is not present in your
plant extract, is chemically stable, ionizes easily, and its retention time does not coincide with that of any analytes (to avoid
ion suppression). For example formononetin or reserpine are
Transportomics for Apocarotenoid Transporters
97
(e.g., 0.5 μg/mL formononetin) to the glass vial.
2. Transfer the glass vial into a shaker with controlled temperature. For heat-stable compounds perform the incubation
through continuous agitation for 30 min at 30–37
C. For
heat-labile compounds perform the incubation at 20–25
C.
3. To remove microsome lipids, transfer 800 μL of eluted samples
in a safe-lock microcentrifuge tube and add 400 μL of chloroform. Vortex for 30 s and shake in a homogenizer, for example
use a Mixer Mill MM 400 (Retsch) or similar for 5 min at
20 Hz frequency. Add 200 μL ultrapure water to separate
phases, vortex and centrifuge at 20,000 Â g for 20 min.
4. Recover 800 μL of the upper phase and transfer into a
new tube.
5. Dry the sample in a vacuum concentrator and resuspend it in
80 μL of 50% (v/v) methanol; centrifuge at 20,000 Â g for
10 min to remove precipitates.
6. Analyze an aliquot of the supernatant by LC-PDA-HRMS (see
Note 8).
4 Notes
1. Do not autoclave solutions described in Subheadings 2.3 and
2.4; store at 4
C for few weeks.
2. Select an adequate yeast strain to perform the transport assays.
For example, S. cerevisiae contains five vacuolar localized
ABCC members (Ycf1p, Bpt1p, Ybt1p/Bat1p, Nft1p, and
Vmr1p) [22]. So, in case of studying a plant ABCC transporter
it is advisable to test yeast strains mutated in one or multiple
ABCCs present in S. cerevisiae and perform transport assays
using microsomes purified from yeasts that heterologous
express the transporter of interest.
3. A critical point to obtain intact and functional yeast microsomes is the yeast growth. Using our experimental conditions,
we obtained good results when OD600 is 3–3.5 after O/N
growth. Furthermore it is suggested to test the physiological
intactness of the isolated microsomes before transport assays.
This can be performed by the vesicles lumen acidification
method as described in [23].
4. Choose as Internal standard (IS) a compound that has similar
chemical properties to your analytes, is not present in your
plant extract, is chemically stable, ionizes easily, and its retention time does not coincide with that of any analytes (to avoid
ion suppression). For example formononetin or reserpine are
Transportomics for Apocarotenoid Transporters
97