Phosphoproteomics-identified ERF110 affects ... - Plant Physiology

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The frozen Arabidopsis plant tissues (4 g) were ground to fine powder with an
ice-cold mortar and pestle. The fine tissue powder was extracted with 12 mL UEB
protein extraction buffer containing 150 mM Tris-HCl pH 7.6, 8 M urea, 0.1%
sodium dodecyl sulfate (SDS), 1.2 % triton X-100, 5 mM ascorbic acid, 50 mM DTT,
20 mM ethylenediaminetetraacetic acid (EDTA), 20 mM ethylene glycol tetraacetic
acid (EGTA), 50 mM NaF, 1% glycerol 2-phosphate, 1 mM PMSF, 0.5% phosphatase
inhibitor cocktail 2 (Sigma P5726), 0.5% protease inhibitor (complete EDTA free,
Roche) and 2% polyvinyl polypyrrolidone (PVPP) (Guo and Li, 2011). The extract
was centrifuged at 110 000 × g for 2 h at 10 °C to remove cell debris. The total
protein supernatant fraction was precipitated with three volumes of pre-cold
acetone:methanol (12:1). The protein pellet was collected by centrifugation and
re-suspended in protein resuspension buffer (50 mM Tris-HCl pH 6.8, 8 M urea, 50
mM DTT, 20 mM EDTA, 2% SDS). The resulting protein extraction was then used
either for western blot analysis or purification of over-expressed ERF110 protein from
plant cells to determine in-vivo phosphorylation sites and phosphorylation occupancy
(Raqu or Risf; Li et al., 2012).
The over-expressed ERF110 was purified by tandem affinity purification as described
previously (Li et al., 2012). The protein extract underwent a standard Ni 2+ -NTA beads
(Qiagen) purification procedure. Proteins were eluted three times with 1 mL buffer B
(8 M urea, 200 mM NaCl, 10 mM sodium phosphate, 0.2% SDS, 100 mM Tris, 250
mM immidazole) and loaded onto immobilized streptavidin magnetic beads
(Invitrogen). The protein-beads mixture was incubated overnight at room temperature
and washed three times with 1 mL buffer C (8 M urea, 200 mM NaCl, 0.2% SDS, 100
mM Tris, pH 8.0). Biotin-labeled protein was eluted using 1× SDS loading buffer
containing 30 mM D-biotin at 96 ºC for 15 min and ice-chilled quickly. The resulting
ERF110 fusion proteins were separated on SDS–polyacrylamide gel electrophoresis
(PAGE) and the corresponding band of ERF110 was sliced out, followed by a
standard in-gel trypsin digestion protocol. The digested peptides were desalted with