2. At least 100 pestle strokes per sample are performed for complete homogenization (see Note 5).
3. The homogenate is filtered through 4 layers of miracloth into a
50 mL falcon tube (see Note 5).
4. The homogenate is centrifuged at 7500 g at 4
C for 15 min.
5. The supernatant is carefully transferred into a new tube and
centrifuged again at 48,000 g at 4
C for 75 min.
6. The microsomal (MF) pellet is resuspended in 150–200 μL
UTU on ice (see Note 6).
7. Protein concentration is measured with Bradford assay. 1 μL
MF is mixed with 24 μL deionized water and 200 μL Bradford
reagent. The mixture is incubated at room temperature in
darkness for 10 min, and absorbance is measured at 590 nm.
8. 100 μg MF are aliquoted into Eppendorf tubes and stored at
À80
C (see Note 7).
3.2 In Solution
Trypsin Digestion
(Timing ~17 h)
1. 2 μL of reduction buffer is added (1 μL reduction buffer for
every 50 μg protein) into the sample and the mixture is incubated at room temperature for 30 min.
2. 2 μL of alkylation buffer (1 μL alkylation buffer for every 50 μg
protein) is added into the sample and the mixture is incubated
at room temperature in darkness for 20 min (see Note 8).
3. 2 μL of Lys-C (1 μL Lys-C for every 50 μg protein) is added
into the sample and the mixture is incubated at room temperature for 3 h.
4. The sample is diluted with 4 volumes of 10 mM Tris–HCl
(pH 8.0).
5. 2 μL of trypsin (1 μL trypsin for every 50 μg protein) is added
into the sample and the mixture is incubated at 37
C
overnight.
6. Digestion is quenched by acidification with TFA to 0.2%
(vol/vol).
7. Digested peptides are dried with speed vacuum concentrator at
30
C (see Note 9).
3.3 TiO 2
Phosphopeptide
Enrichment
(Timing ~2 h)
1. TiO 2 beads (10:1 ratio TiO 2 beads/protein) are added into
100 μL methanol (Gradient grade, !99.9%) and centrifuged at
2500 g at room temperature for 2 min. The supernatant is then
carefully removed and discarded.
2. TiO 2 beads are subsequently washed once with 100 μL of
elution buffer for 10 min with vortex mixing and centrifuged
at 2500 g at room temperature for 2 min. The supernatant is
then discarded. TiO 2 beads are equilibrated with 100 μl of
resuspension buffer for 1 min and centrifuged at 2500 g at
Plant Membranes Phosphoproteome
445
3. The homogenate is filtered through 4 layers of miracloth into a
50 mL falcon tube (see Note 5).
4. The homogenate is centrifuged at 7500 g at 4
C for 15 min.
5. The supernatant is carefully transferred into a new tube and
centrifuged again at 48,000 g at 4
C for 75 min.
6. The microsomal (MF) pellet is resuspended in 150–200 μL
UTU on ice (see Note 6).
7. Protein concentration is measured with Bradford assay. 1 μL
MF is mixed with 24 μL deionized water and 200 μL Bradford
reagent. The mixture is incubated at room temperature in
darkness for 10 min, and absorbance is measured at 590 nm.
8. 100 μg MF are aliquoted into Eppendorf tubes and stored at
À80
C (see Note 7).
3.2 In Solution
Trypsin Digestion
(Timing ~17 h)
1. 2 μL of reduction buffer is added (1 μL reduction buffer for
every 50 μg protein) into the sample and the mixture is incubated at room temperature for 30 min.
2. 2 μL of alkylation buffer (1 μL alkylation buffer for every 50 μg
protein) is added into the sample and the mixture is incubated
at room temperature in darkness for 20 min (see Note 8).
3. 2 μL of Lys-C (1 μL Lys-C for every 50 μg protein) is added
into the sample and the mixture is incubated at room temperature for 3 h.
4. The sample is diluted with 4 volumes of 10 mM Tris–HCl
(pH 8.0).
5. 2 μL of trypsin (1 μL trypsin for every 50 μg protein) is added
into the sample and the mixture is incubated at 37
C
overnight.
6. Digestion is quenched by acidification with TFA to 0.2%
(vol/vol).
7. Digested peptides are dried with speed vacuum concentrator at
30
C (see Note 9).
3.3 TiO 2
Phosphopeptide
Enrichment
(Timing ~2 h)
1. TiO 2 beads (10:1 ratio TiO 2 beads/protein) are added into
100 μL methanol (Gradient grade, !99.9%) and centrifuged at
2500 g at room temperature for 2 min. The supernatant is then
carefully removed and discarded.
2. TiO 2 beads are subsequently washed once with 100 μL of
elution buffer for 10 min with vortex mixing and centrifuged
at 2500 g at room temperature for 2 min. The supernatant is
then discarded. TiO 2 beads are equilibrated with 100 μl of
resuspension buffer for 1 min and centrifuged at 2500 g at
Plant Membranes Phosphoproteome
445