Phosphoproteomics-identified ERF110 affects ... - Plant Physiology

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PCR System for qRT-PCR was purchased from Promega (Madison, WI, USA). M/S
basal salt mixture, sucrose, trifluoroacetic acid (TFA) and other chemicals were
purchased from Sigma–Aldrich (St. Louis, MO, USA).
In vitro kinase assay and PTM site quantification using iTRAQ labeling and
MS/MS analysis
Both the in vitro kinase assay and synthetic peptide purification were performed
according to a previously described method (Li et al., 2009). Total cellular proteins
were extracted from the frozen fine powders of Arabidopsis tissues using a kinase
extraction buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl, 1% triton X-100,
2.5 mM sodium pyrophosphate, 1 mM sodium fluoride, 1 mM sodium orthovanadate,
1 mM sodium molybdate, 1 mM glycerol-2-phosphate and 1 mM
phenylmethanesulfonylfluoride (PMSF) protease inhibitors mix (Roche, Switzerland).
Plant cells were mixed with the kinase buffer at a ratio of 1:3 (W:V), which was
incubated on ice for an additional 10 min. The plant kinase extract was then
centrifuged for 10 min at 14000 × g at 4 °C to remove cell debris. Plant kinase
extracts were activated by adding a 1/4 volume of kinase assay buffer [45% glycerol,
2.5 mM adenosine triphosphate (ATP), 50 mM MgCl2 125 µg/mL bovine serum
albumin (BSA)]. Substrate peptides were added at a concentration of 10 µM. The
kinase-substrate mixture was incubated at 30 °C for 1 h. Peptides were purified using
Ni 2+ -NTA agarose beads (Qiagen) according to the manufacturer’s instructions.
Following overnight digestion using trypsin at 37 °C, the purified peptides were
desalted with C18 Zip-tip columns and re-suspended in 0.1% trifluoroacetic acid
(TFA) for liquid chromatography (LC)-MS/MS analysis.
In the iTRAQ experiment, the purified ERF110 peptides containing Ser62
phosphosite (Supplementary Table 1) were first labeled with iTRAQ 4-Plex labeling
reagent (Applied Biosystems) following the kinase assay. The isotope-labeled
phosphopeptides were purified with TiO2 beads according to the manufacturer’s
instructions and desalted with C18 Zip-tip columns and re-suspended in 0.1% TFA
for MALDI-MS/MS analysis using a Bruker Autoflex III MALDI TOF/TOF MS
/Dionex. The iTRAQ experiment was repeated on the same plant at least twice with a
reciprocal labeling of iTRAQ chemicals to exclude the difference brought about by
the differential labeling process. The ion intensity of the reporter ion of the air-treated