3. It is recommended to freshly prepare this buffer before use.
4. We use the Empore discs of C18 or C8 material (Life Technologies) and use a blunt dermatological needle to punch out
small discs that can be stuffed into a 200 μL pipette tip. Details
on stage-tip manufacturing are described elsewhere [14]. In
general, desalting over C18 can, in principle, also be done
using alternative products such as ZIP-TIPs or C18 cartridges.
5. Plant materials are harvested with aluminum foil and broken
immediately when removed from the À80
C freezer without
the use of liquid nitrogen. We use Teflon-glass grinders
(Dounce homogenizer) for maintaining the membrane integrity and increasing the efficiency of membrane fraction
extraction.
6. Resuspension of the MF should be very slow to avoid many
bubbles. At this step, the MF can be stored at À20
C for shorttime storage or À80
C for long-time storage.
7. We use 100–200 μg MF for phosphopeptide enrichment since
1 g starting plant materials are applied. If you have more
starting materials and more MF isolated, you may use more
MF for trypsin digestion and phosphopeptide enrichment.
8. IAA is light sensitive; this step should be performed in
darkness.
9. Digested samples can be stored at À20
C for short-time
storage or À80
C for long-time storage.
10. If more than 200 μg MF are used, then 400 μL or more
resuspension buffer should be used.
11. Dissolved peptides from step 3 are suggested to be spun down
and supernatant should be transferred carefully to the
Eppendorf tube.
12. The supernatant contains the un-phosphorylated peptides.
Here, we suggest to keep the supernatant (in storage) in case
it is necessary to compare between un-phosphorylated peptides
and phosphorylated peptides from the same sample. These
supernatant should be stored at À80
C for long-time storage.
13. A very basic solution can induce the loss of phosphate groups
from peptides; it is crucial to add acidified buffer immediately.
Enriched peptide samples can be stored at À20
C for shorttime storage or–80
C for long-time storage.
14. A C18 analytical capillary column is used for eluting peptides
and spaying them directly into the mass spectrometer (EASYSpray analytical column, 25 cm  75 μm inner diameter). The
settings may need to be adjusted for measurements when
alternative LC and MS instruments are chosen.
Plant Membranes Phosphoproteome
449
4. We use the Empore discs of C18 or C8 material (Life Technologies) and use a blunt dermatological needle to punch out
small discs that can be stuffed into a 200 μL pipette tip. Details
on stage-tip manufacturing are described elsewhere [14]. In
general, desalting over C18 can, in principle, also be done
using alternative products such as ZIP-TIPs or C18 cartridges.
5. Plant materials are harvested with aluminum foil and broken
immediately when removed from the À80
C freezer without
the use of liquid nitrogen. We use Teflon-glass grinders
(Dounce homogenizer) for maintaining the membrane integrity and increasing the efficiency of membrane fraction
extraction.
6. Resuspension of the MF should be very slow to avoid many
bubbles. At this step, the MF can be stored at À20
C for shorttime storage or À80
C for long-time storage.
7. We use 100–200 μg MF for phosphopeptide enrichment since
1 g starting plant materials are applied. If you have more
starting materials and more MF isolated, you may use more
MF for trypsin digestion and phosphopeptide enrichment.
8. IAA is light sensitive; this step should be performed in
darkness.
9. Digested samples can be stored at À20
C for short-time
storage or À80
C for long-time storage.
10. If more than 200 μg MF are used, then 400 μL or more
resuspension buffer should be used.
11. Dissolved peptides from step 3 are suggested to be spun down
and supernatant should be transferred carefully to the
Eppendorf tube.
12. The supernatant contains the un-phosphorylated peptides.
Here, we suggest to keep the supernatant (in storage) in case
it is necessary to compare between un-phosphorylated peptides
and phosphorylated peptides from the same sample. These
supernatant should be stored at À80
C for long-time storage.
13. A very basic solution can induce the loss of phosphate groups
from peptides; it is crucial to add acidified buffer immediately.
Enriched peptide samples can be stored at À20
C for shorttime storage or–80
C for long-time storage.
14. A C18 analytical capillary column is used for eluting peptides
and spaying them directly into the mass spectrometer (EASYSpray analytical column, 25 cm  75 μm inner diameter). The
settings may need to be adjusted for measurements when
alternative LC and MS instruments are chosen.
Plant Membranes Phosphoproteome
449