Proteome Analysis
Extraction and digestion - Samples were randomized before processing to avoid batch effects. Frozen rosettes were ground under liquid N2. Proteins were extracted from 100 mg of frozen powder per sample by adding 150 µl of extraction buffer (30 mM Tris-HCl pH 8.0, 4% SDS). Tubes were incubated in a shaker (Eppendorf) at 4ºC at 1400 rpm for 30 min. Samples were centrifuged at 16000 g and 4ºC for 30 min and the supernatant was transferred to a new tube. Protein concentration was estimated based on Bradford (Bradford, 1976) using the Bio-Rad Protein Assay reagent. Subsequently, DTT was added to a final concentration of 50 mM and proteins were reduced for 30 min on ice. For digestion, 140 µg of proteins were processed following the FASP method (Wisniewski, Zougman, Nagaraj, & Mann, 2009). Peptides were desalted using SPE C18 columns (Finisterre) and dried down in a SpeedVac concentrator.
Peptide fractionation - To increase proteome coverage, peptide samples were fractionated by hydrophilic interaction chromatography (HILIC) on an Agilent 1200 series HPLC system with a YMC‑Pack Polyamine II 250 x 3.0 mm size column with 5 μm particle size and 120 Å pore size. Samples were dissolved in 100 μl Buffer A (75% ACN, 8 mM KH2PO4, pH 4.5) and separated with Buffer B (5% ACN, 100 mM KH2PO4, pH 4.5) at a flow rate of 500 µl/min with the following gradient: 0‑7.5 min, 0% B; 7.5‑37.5 min, 0‑50% B; 37.5‑42.5 min, 50‑100% B; 42.5‑47.5 min, 100% B. Following the separation the column was washed with 100% buffer A and re-equilibrated for 60 min. For each sample, the 27 automatically collected fractions were pooled into five fractions that were subsequently dried down in a SpeedVac concentrator. Each sample was then dissolved in 200 µl of 3% ACN, 0.1% TFA, desalted on SPE C18 columns (Finisterre) and again dried in a SpeedVac concentrator.
LC-MS analysis - Mass spectrometry queues were arranged to process comparable fractions in the same batch, with sample order randomized within each batch. Peptide samples were dissolved in 20 µl 3% ACN, 0.1% FA and spiked with internal retention time (iRT) standards (Biognosys) for chromatography quality control. LC-MS/MS shotgun analyses were performed on a Thermo Orbitrap Fusion instrument coupled to an Eksigent NanoLC Ultra (Sciex). Samples were separated on a self‑packed reverse‑phase column (75 µm x 150 mm) with C18 material (ReproSil-Pur, C18, 120 Å, AQ, 1.9 µm, Dr. Maisch GmbH). The column was equilibrated with 100% solvent A (0.1% FA in water). Peptides were eluted using the following gradient of solvent B (0.1% FA in ACN) at a flow rate of 0.3 µl/min: 0‑50 min: 3‑25% B, 50‑60 min: 25‑35% B, 60‑70 min: 35-97% B, 70‑80 min: 97% B, 80‑85 min: 2% B. Mass spectra were acquired in a data-dependent manner. All precursor signals were recorded in the Orbitrap using quadrupole transmission in the mass range of 300‑1500 m/z. Spectra were recorded with a resolution of 120000 (FWHM) at 200 m/z, a target value of 4e5 and the maximum cycle time set to 3 s. Data dependent MS/MS were recorded in the linear ion trap using quadrupole isolation with a window of 1.6 Da and higher-energy collisional dissociation (HCD) fragmentation with 30% fragmentation energy. The ion trap was operated in rapid scan mode with a target value of 1E4 and a maximum injection time of 250 ms. Precursor signals were selected for fragmentation with a charge state from + 2 to + 7 and a signal intensity of at least 5e3. A dynamic exclusion list was used for 30 s and maximum parallelizing ion injections was activated. The mass spectrometry proteomics data were handled using the local laboratory information management system (LIMS) (Türker et al., 2010)

Phosphoproteome Analysis

Extraction – Whole rosette tissue from each time point was harvested and ground under liquid N2. From each biological replicate 200 mg of ground leaf material was weighed out under liquid N2. In addition to each biological replicate, 200 mg of samples containing equal weighted parts of each biological replicate and time-point were created as a reference sample (gold-standard) for downstream dimethyl labeling. All proteins were extracted in a 250 µl solution of 50 mM HEPES pH 8.0, 6 M urea, 2 M thiourea, 100 mM NaCl, 10 mM EDTA, 2 mM NaOV, 5 mM NaF, 50 µg/mL PhosSTOP (Roche). Samples were shaken at room temperature for 30 min at 1000 g with vortexing every 10 min. Extracts were then brought to pH 8.0 using triethylammonium bicarbonate (TEAB). Protein extracts were then reduced for 30 min with 10 mM DTT, followed by alkylation with 30 mM iodoacetamide for 1 h. Extracts were clarified to separate soluble and insoluble fractions. The insoluble fraction was re-suspended in 300 µL 60:40 buffer containing 60% MeOH: 40% 50 mM TEAB pH 8.0 followed by shaking at 1000 rpm (Eppendorf tabletop) for 2.5 h. The protein concentration of the soluble fraction was then measured using the Bradford protein assay (Bradford, 1976). An amount of 1 mg of soluble protein from each sample was then diluted with 1 vol. of 50 mM TEAB and then water was added to a total volume of 1.2 ml and a final urea/thiourea concentration of 1.2 M. The soluble fraction was then digested for 20 h at 37ºC using a 1:50 ratio of trypsin (Promega) to extracted protein while gently shaking. Each insoluble fraction was digested by 0.5 µg chymotrypsin and 1 µg trypsin at 37ºC for 20 h shaking at 600 rpm (Eppendorf tabletop). Digestion reactions were stopped using TFA to a final concentration of 0.5%. The insoluble fractions were centrifuged for 10 min at 20000 g at room temperature and the supernatant removed. The supernatant was then dried and re-suspended in desalting buffer comprised of 3% ACN / 0.1% TFA. The soluble fraction and the supernatant from the insoluble fraction were desalted using SPE C18 columns (Finisterre) and dried in a SpeedVac concentrator.
Dimethyl labeling and phosphopeptide enrichment - Total peptide fractions from each experimental (light label) and gold-standard (heavy label) sample were labeled according to Boersema et al.,(Boersema, Raijmakers, Lemeer, Mohammed, & Heck, 2009). Heavy and light samples were then mixed 1:1 and desalted prior to phosphopeptide enrichment using TiO2. Phosphopeptide enrichment was performed using TiO2 heavy and light dimethyl-labelled phosphopeptides as previously described (Zhou et al., 2011).
LC-MS - Phosphorylated peptide samples were analyzed using a Q Exactive Orbitrap mass spectrometer (Thermo Scientific). Dissolved samples were injected using an Easy-nLC 1000 system (Thermo Scientific) and separated on a self-made reverse-phase column (75 µm x 150 mm) packed with C18 material (ReproSil-Pur, C18, 120 Å, AQ, 1.9 µm, Dr. Maisch GmbH). The column was equilibrated with 100% solvent A (0.1% formic acid (FA) in water). Peptides were eluted using the following gradient of solvent B (0.1% FA in ACN): 0-120 min, 0-35% B, 120-122 min, 35-95% B at a flow rate of 0.3 µl/min. High accuracy mass spectra were acquired in data-depended acquisition mode. All precursor signals were recorded in a mass range of 300-1700 m/z and a resolution of 70000 at 200 m/z. The maximum accumulation time for a target value of 3e6 was set to 120 ms. Up to 12 data dependent MS/MS were recorded using quadrupole isolation with a window of 2 Da and HCD fragmentation with 28% fragmentation energy. A target value of 1e6 was set for MS/MS using a maximum injection time of 250 ms and a resolution of 70000 at 200 m/z. Precursor signals were selected for fragmentation with charge states from +2 to +7 and a signal intensity of at least 1e5. All precursor signals selected for MS/MS were dynamically excluded for 30 s.