SUPPORTING INFORMATION APPENDIX Comparative phosphoproteome profiling reveals a function of the STN8 kinase in the fine- tuning of cyclic electron flow (CEF) Sonja Reiland, Anne Endler, Adrian Willig, Katja Baerenfaller, Jonas Grossmann, Bertran Gerrits, Dorothea Rutishauser, Giovanni Finazzi, Wilhelm Gruissem, Jean David Rochaix and Sacha Baginsky, Supplemental Materials and Methods Supplemental Figures Supplemental Tables SUPPLEMENTAL TEXT MATERIALS AND METHODS Plant material and growth conditions. Arabidopsis thaliana Col0 and stn8 (SALK 060869) seedlings were grown on soil under short day conditions in a controlled environment chamber (8 h light/16 h dark, 100 μE m-2 s-1). Plants were harvested after 6 weeks, 3 h after the start of the light and immediately frozen in liquid nitrogen grinded and subsequently stored at -80°C until further analyses. Protein extraction Protein extraction was done in two steps. First, soluble proteins were extracted from frozen and grinded plant material by adding 40 mM Tris-HCl pH8, 5 mM MgCl, 1 mM DTT, inhibitor cocktail for proteases (EDTA free, Roche) and phosphatases (phosSTOP, Roche). Second, 40 mM Tris, 4% SDS, 40 mM DTT was added to extract membrane-associated and integral membrane proteins. Soluble proteins were precipitated by adding 5 volumes of ice cold 80% acetone and 3 h incubation at -20°C. Membrane proteins were precipitated by methanol and chloroform (1). Finally, both protein pellets were resolved separately in a small volume of resuspension buffer (20 mM Tris-HCl pH 8.3, 3 mM EDTA, 8 M urea). For tryptic digestion the protein solution was diluted to 1 M urea by adding 20 mM Tris-HCl pH 8.3, 3 mM EDTA. In solution tryptic protein digest Before tryptic digest, cysteine residues were reduced with 10 mM DTT for 45 min at 50°C and alkylated with 50 mM iodoacetamide for 1 hr at room temperature in the dark. Trypsin (Promega, sequencing grade) was added in a ratio of 1:20 and incubated over night at 37 °C. Fractionation of Peptides by Strong Cation Exchange Chromatography (SCX) Peptides were desalted using Sep-Pak reverse-phase cartridges (Waters, UK), dissolved in buffer A (10 mM KH2PO4 pH 2.6 in 25% acetonitrile) and loaded onto a 4.6 x 200 mm polySULFOETHYL aspartamide A column (PolyLC, USA) on an Agilent HP1100 binary HPLC system. Peptides were eluted with an increasing KCl gradient (10-40 min 0-30% buffer B, 40-60 min 30-100% buffer B, buffer B: 10 mM KH2PO4 pH 2.6, 350 mM KCl in 25% acetonitrile). The eluate was fractionated into four fractions and desalted with Sep-Pak reverse-phase cartridges (Waters, UK). Immobilized Metal Ion Affinity Chromatography (IMAC) Chelating Sepharose Fast Flow beads (GE Healthcare) were charged four times with 0.1 M FeCl3 freshly prepared solution and washed four times with washing buffer (74:25:1 water:acetonitrile:acetic acid). Desalted peptides were acidified with 0.1% TFA in 25% acetonitrile, applied to 40 µl of 25% bead slurry and incubated for 30 min at room temperature. Samples were washed five times with washing buffer and once with water. Phosphopeptides were eluted by adding 30 µl of 100 mM sodium phosphate buffer pH 8.9. The pH of all samples was adjusted to 3 by adding drops of 10% TFA followed by desalting and concentrating samples using ZipTips (C18, Millipore). Titanium Dioxide (TiO2) Affinity Chromatography Phosphopeptides were enriched using TiO2 affinity chromatography as described by Bodenmiller and colleagues (2) with minor modifications. Peptides were desalted and dissolved in phthalic acid solution (80% ACN, 2.5% TFA, 0.13 M phthalic acid). The peptide mixture was incubated with 0.3 mg TiO2 (GL Science, Saitama, Japan) for 30 m in closed Mobicol spin columns. After washing twice with phthalic acid solution, twice with 80% ACN, 0.1% TFA, once with 0.1% TFA and finally again once with 80% ACN, 0.1% TFA, peptides were eluted with 0.3 M NH4OH and dried in a speed vac. Before mass spectrometric analysis samples were desalted using ZipTips (C18, Millipore). Analysis by LC-ESI-MS/MS Phosphopeptide Analysis was performed with an LTQ-Orbitrap as described previously (3). Dried peptides were resuspended in 5% ACN, 0.1% formic acid and analyzed on a LTQ-Orbitrap mass spectrometer (ThermoFischer Scientific, Bremen, Germany) interfaced with a nanoelectrospray ion source. Peptides were separated using an Eksigent nano LC system (Eksigent Technologies, Dublin, CA, USA), equipped with an 11 cm fused silica emitter, 75 µm inner diameter (BGB Analytik, Böckten, Switzerland), packed in-house with a Magic C18 AQ 3 µm resin (Michrom BioResources, Auburn, CA, USA). Peptides were loaded from a cooled (10°C) Spark Holland auto sampler and separated using an ACN/water solvent system containing 0.1% formic acid at a flow rate of 200 nl/min. Peptide mixtures were separated by gradient elution from 3 to 35% ACN in 90 min. Up to 5 data dependent MS/MS spectra were acquired in the linear ion trap for each FT-MS spectral acquisition range, the latter acquired at 60,000 FWHM nominal resolution settings with an overall cycle time of approximately 1 s. Dynamic exclusion was switched on entailing that up to 500 m/z ± 20 ppm values were excluded from tandem MS for 120 s. For injection control the automatic gain control was set to 5e5 for full FTMS and to 1e4 for linear ion trap MS2. The instrument was calibrated externally according to manufacturer’s instructions. The samples were acquired using internal lock mass calibration on m/z 429.088735 and 445.120025. Data Analysis and MS/MS spectra interpretation MS/MS spectra were searched with Mascot (Matrix Science, London, UK) version 2.2.04 against the TAIR9 (The Arabidopsis Information Resource) protein database (download on June 29th, 2009) with concatenated decoy database supplemented with contaminants (67’079 entries). The search parameters were: mass = monoisotopic, requirement for tryptic ends, 2 missed cleavages allowed, precursor ion tolerance = +/- 10 ppm, fragment ion tolerance = +/- 0.8 Da, allowing for 2+ and 3+ charged peptides, static modifications of cysteine (C, PSI-MOD name: iodoacetamide derivative, mono Δ = 57.021464) and variable modifications of methionine (M, PSI-MOD name: oxidation, mono Δ = 15.9949), serine (S, PSI-MOD name: O-phosphorylated L-serine, mono Δ = 79.9663), threonine (T, PSI-MOD name: O-phosphorylated L-threonine, mono Δ = 79.9663), tyrosine (Y, PSI-MOD name: 4’-phospho-L-tyrosine, mono Δ = 79.9663), lysine and N-terminus (K and N-term, PSI-MOD name: acetylated residue, mono Δ = 42.0106). Identifications were accepted with a Mascot ion score > 30 and a Mascot expect value < 0.015 (4). A normalized delta ion score (ΔI) was calculated for phosphopeptides for which the only difference between the rank 1 and the rank 2 hit was the phosphorylation position. ΔI was calculated by taking the difference of the two top ranking ion scores and dividing that difference by the ion score of the first ranking phosphopeptide. Phosphorylation site assignments with ΔI ≥ 0.4 were accepted (3, 5, 6). All peptide assignments except those of contaminants were filtered for ambiguity and the peptides matching to several proteins were excluded from further analysis. This does not apply to different splice variants of the same protein or to different loci sharing exactly the same sequence. After database upload spectrum assignments to decoy database peptides were flagged. From the final data PRIDE 2.1 XML files were created and exported to the PRIDE database (7). The spectrum false discovery rate was calculated by dividing the number of decoy database spectrum assignments by the number of spectrum assignments in the final dataset. Relative quantification by extracted ion chromatograms was achieved by commercial ProGenesis software from Nonlinear Dynamics. Analysis was performed in pairs of LC-MS-runs for wild- type and the corresponding stn8 experiment. The quantitative analysis was done in three biological replicates. Spectroscopic measurements Spectroscopic measurements were performed on intact leaves with a flash spectrophotometer (JTS 10, Biologic France). P700 oxidation kinetics was assessed at 820-870 nm, as previously described (8). Far-red illumination was provided by a LED peaking at 720 nm, filtered through three Wratten filters 55 that block wavelengths shorter than 700 nm. When needed, the + maximum extent of P700 was estimated by imposing a saturating flash of white light on top of the far red, as described in (9,10). Actinic light was provided by a red LED peaking at 620 nm. Membrane potential changes (ECS) were measured under the same actinic illumination used for measurements of the P700 redox changes. They were detected at 520-545 nm, using a white LED source (Luxeon, Lumileds) filtered through appropriate interference filters. This procedure allows deconvoluting the ECS signal from absorption changes associated with redox changes related to electron flow (e.g. the cytochrome b6f complex). The photodiodes were protected from actinic light by a Schott BG 39 filter. To avoid excessive exposure of leaves to light and dark cycles, we limited our analysis to the minimum number of wavelengthsrequired for proper estimation of the different optical parameters. Therefore, since no sustained zeaxanthin synthesis was detected at this low light intensity, spectroscopic changes associated with the generation of a transmembrane potential were not routinely deconvoluted from the zeaxanthin red shift signal (A535, (11)). However, we checked that no major modification of the ECS kinetics profiles occurred when this deconvolution was performed using appropriate wavelengths (505 and 535 nm, e.g. (12)) in some test experiments. In the case of P700 and membrane potential changes, continuous actinic light was transiently switched off while measuring at appropriate wavelengths. Fluorescence was measured with the same apparatus used for spectroscopy.