7. Wash buffer for phosphopeptide enrichment: 80% ACN and
1% TFA. Mix well 8 mL ACN, 0.1 mL TFA, and 1.9 mL
deionized water.
8. Elution buffer for phosphopeptide enrichment: 1% Ammonium hydroxide solution (NH 4 OH, stock concentration
25%). Mix well 0.4 mL 25% ammonium solution and 9.6 mL
deionized water.
9. Acidified buffer: 10% Formic acid. Mix well 1 mL formic acid
and 9 mL deionized water.
10. 2% Trifluoroacetic acid (TFA): Add 2 mL TFA into 98 mL
deionized water, and mix well.
11. Solution A for C18 stage-tip: 0.1% TFA and 5% ACN. Mix well
100 μL TFA and 5 mL ACN into 94.9 mL deionized water.
12. Solution B for C18 stage-tip: 0.1% TFA and 80% ACN. Mix
well 100 μL TFA and 80 mL ACN into 19.9 mL deionized
water.
13. Solvent A for HPLC: 0.5% acetic acid. Mix well TFA, ACN and
water.
14. Solvent B for HPLC: 0.5% acetic acid and 80% ACN. Mix well
500 μL TFA and 80 mL ACN into 19.5 mL HPLC water.
2.2 Other Materials
1. C18 stage-tip: 200 μL tips + two disc containing C18 materials
(see Note 4).
2. Lys-C: sequencing-grade (0.5 μg/μL).
3. Trypsin: sequencing-grade modified (0.5 μg/μL).
4. Titanium dioxide (TiO 2 ) beads: 5 μm.
2.3 Specific
Equipment
1. Nanoflow Easy-nLC 1000 HPLC system (Thermo Scientific,
Germany).
2. Orbitrap hybrid mass spectrometer (Q-Exactive plus, Thermo
Scientific, Germany).
3 Methods
3.1 Preparation of
Microsomal Fraction
(Timing ~2–3 h)
1. A total of 1–1.5 g frozen plant material (fresh weight) for each
of three biological replicates (stored in a À80
C freezer until
needed) are broken into small pieces with a hammer or a pestle
(see Note 5). Then the plant materials are carefully poured into
a 10 cm
3 Teflon-glass grinder and homogenized with HB
buffer. Pieces of plant materials should be incubated into HB
for 10 min on the ice before grinding (see Note 5). See Fig. 1a
for an overview of workflow.
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Lin Xi et al.
1% TFA. Mix well 8 mL ACN, 0.1 mL TFA, and 1.9 mL
deionized water.
8. Elution buffer for phosphopeptide enrichment: 1% Ammonium hydroxide solution (NH 4 OH, stock concentration
25%). Mix well 0.4 mL 25% ammonium solution and 9.6 mL
deionized water.
9. Acidified buffer: 10% Formic acid. Mix well 1 mL formic acid
and 9 mL deionized water.
10. 2% Trifluoroacetic acid (TFA): Add 2 mL TFA into 98 mL
deionized water, and mix well.
11. Solution A for C18 stage-tip: 0.1% TFA and 5% ACN. Mix well
100 μL TFA and 5 mL ACN into 94.9 mL deionized water.
12. Solution B for C18 stage-tip: 0.1% TFA and 80% ACN. Mix
well 100 μL TFA and 80 mL ACN into 19.9 mL deionized
water.
13. Solvent A for HPLC: 0.5% acetic acid. Mix well TFA, ACN and
water.
14. Solvent B for HPLC: 0.5% acetic acid and 80% ACN. Mix well
500 μL TFA and 80 mL ACN into 19.5 mL HPLC water.
2.2 Other Materials
1. C18 stage-tip: 200 μL tips + two disc containing C18 materials
(see Note 4).
2. Lys-C: sequencing-grade (0.5 μg/μL).
3. Trypsin: sequencing-grade modified (0.5 μg/μL).
4. Titanium dioxide (TiO 2 ) beads: 5 μm.
2.3 Specific
Equipment
1. Nanoflow Easy-nLC 1000 HPLC system (Thermo Scientific,
Germany).
2. Orbitrap hybrid mass spectrometer (Q-Exactive plus, Thermo
Scientific, Germany).
3 Methods
3.1 Preparation of
Microsomal Fraction
(Timing ~2–3 h)
1. A total of 1–1.5 g frozen plant material (fresh weight) for each
of three biological replicates (stored in a À80
C freezer until
needed) are broken into small pieces with a hammer or a pestle
(see Note 5). Then the plant materials are carefully poured into
a 10 cm
3 Teflon-glass grinder and homogenized with HB
buffer. Pieces of plant materials should be incubated into HB
for 10 min on the ice before grinding (see Note 5). See Fig. 1a
for an overview of workflow.
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Lin Xi et al.