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pmic7205-sup-0001-TableS1.xls1814KTable S1: Identified phosphopeptides from the SILAC shotgun experiment. In two biological SILAC replicates yeast cells were stressed for 5 min with high osmolarity. SILAC2 was measured with three different methods. Unique phosphopeptides identified in these runs are listed in a combined list with the averaged ratio L/H. Cells are marked in blue if the peptide was identified in the corresponding run. SEQUEST-modification, XCorr, number of missed cleavages, Δppm, pRS probability and H/L ratio of all unique phosphopeptides are listed for each experiment separately.
pmic7205-sup-0002-TableS2.xls85KTable S2: Confidently localized phosphorylation sites of Pan1. Pan1-phosphopeptides from all experiments (SILAC and iTRAQ) are listed. List is divided into phosphopeptides with a phosphorylation site probability of >95% and of 75% – 95% according to PhosphoRS 2.0.
pmic7205-sup-0003-TableS3.xls33KTable S3: Non localized phosphorylation sites of Pan1. Pan1-phosphopeptides from all experiments (SILAC and iTRAQ) with a phosphorylation site probability of <75% are listed.
pmic7205-sup-0004-TableS4.xls369KTable S4: SILAC quantification of Pan1 peptides in wild type. Search results from 4 biological replicates are listed. Shown are the averages of normalized stress induction ratios.
pmic7205-sup-0005-TableS5.xls289KTable S5: SILAC quantification of Pan1 peptides in hog1Δ. Search results from 3 biological replicates are listed. Shown are the averages of normalized stress induction ratios.
pmic7205-sup-0006-TableS6.xls218KTable S6: SILAC quantification of Pan1 peptides in hog1Δ kss1Δ. Search results from 2 biological replicates are listed. Shown are the averages of normalized stress induction ratios.
pmic7205-sup-0007-TableS7.xls288KTable S7: iTRAQ quantification of Pan1 peptides in wild type. Search result from a single run. Shown are the averages of normalized stress induction ratios.
pmic7205-sup-0008-TableS8.xls33KTable S8: Comparison of quantitative MS strategies: SRM data. Peak areas of transitions (listed in Supplemental Figure 2C) for phosphorylation sites S1003 and T1225 have been determined for the light and heavy labeled peptides. Stress induction ratios (blue) have been normalized with AQUA-peptide (factor indicated in green) and corrected Arg-Pro conversion (red).
pmic7205-sup-0009-TableS9.xls83KTable S9: Sample preparation strategies for SRM. Peak areas of transitions (listed in Supplemental Figure 2C) for S1003 and T1225 are listed. Stress induction ratios (blue) have been normalized with AQUA-peptide and three reference peptides of Pan1 (factor indicated in green) except for WCE TiO2 samples. Transitions of reference peptides from WCE TiO2 are indicated on the right and labeled with “$” and “$$”.
pmic7205-sup-0010-TableS10.xls67KTable S10: Phosphorylation kinetics by SRM measurements. Peak areas of transitions (listed in Supplemental Figure 2C) for S1003 and T1225 are listed. Stress induction ratios (blue) have been normalized with AQUA-peptide and three reference peptides of Pan1 (factor indicated in green). Time point 0 min has been set to 1.
pmic7205-sup-0011-TableS11.xls129KTable S11: SRM measurements for Hog1-inhibitor studies. Peak areas of transitions (listed in Supplemental Figure 2C) for S1003 and T1225 are listed. Stress induction ratios (blue) have been normalized with AQUA-peptide and three reference peptides of Pan1 (factor indicated in green). Time point 0 min has been set to 1.
pmic7205-sup-0012-TableS12.xls26KTable S12: Normalization and correction factors for Arg-Pro conversion. Normalization factors were calculated for each biological sample based on unique peptides containing no phosphorylation and no proline. The correction factors for Arg-Pro-conversion was determined by the H:L ratio of unphosphorylated peptides containing 1 proline and were extrapolated for peptides containing more than 1 proline.
pmic7205-sup-0013-TableS13.xls19KTable S13: Strains and plasmids used in this study.
pmic7205-sup-0014-FigureS1.tif2354KFigure S1: Performance of the HTBeaq-tag. A: HTBeaq contains a quantification peptide sequence (AQUA), one additional 6xHis tag and two additional TEV cleavage sites. Modifications are highlighted by red boxes. The 158 amino acid sequence is suitable for tagging yeast proteins under the control of the endogenous promoter. B: Optimized protein purification protocol that allows tandem affinity purification of proteins under denaturing conditions. Alternatively a one-step purification using STRP-Agarose as the sole affinity resin can be performed. Purified proteins can be directly digested from the affinity resins and phosphopeptides can be further enriched via TiO2 if necessary before the final analysis by reverse phase nano-HPLC-MS. C: Comparison of HPLC UV chromatograms (λ = 214nm) of either His tag purified (blue), biotinylation tag purified (red), or tandem purified (green) Pan1-HTBeaq. RT: retention time. D: SRM transitions of phosphopeptide SSS#PSYSQFK of Pan1 (S1003) obtained from the three differently purified Pan1-HTBeaq samples. Singly and doubly charged C-terminal y-ions are indicated. NL: neutral loss. Peaks automatically selected for quantification by the software are marked with “Area”. Inset on the left shows SRM transitions of the Ni2+-Sepharose purified sample in higher resolution. Actual peak of peptide SSS#PSYSQFK is marked with an arrow. Inset on the right shows SRM transitions obtained with a synthetic peptide. E: Comparison of different strategies for sample preparation for SRM. Ratios (stressed/unstressed) obtained for phosphopeptides SSS#PSYSQFK (S1003) or SVHAAVT#PAAGK (T1225) and the corresponding unphosphorylated peptides are indicated. SRM measurements have been corrected for proline conversation (correction value was obtained from SILAC quantification Supplemental Table S12).
pmic7205-sup-0015-FigureS2.tif2293KFigure S2: SRM analysis of Pan1 phosphopeptides. A: SRM transitions of phosphopeptides SVHAAVT#PAAGK (T#1225) and SSS#PSYSQFK (S#1003) and of corresponding unphosphorylated peptides shown in Figure 5B. SRM transitions of the quantification peptide embedded in the HTBeaq tag is labeled as AQUA. Pan1-HTBeaq samples were obtained by affinity purification from cultures that have been treated with 0.5 M NaCl for times indicated. Singly and doubly charged C-terminal y-ions are indicated. NL: loss of phosphoric acid. WL: loss of water. B: Patterns and retention times of SRM transitions of peptides obtained from affinity purified Pan1-HTBeaq fit those of corresponding synthetic peptides. SRM transition peaks of a representative sample (sample: wild type + DMSO (mock), osmostressed for 10 minutes) are shown. Peaks automatically selected for quantification by the software are marked with “Area”. C: Detailed list of peptides used in our SRM analysis. m/z values of precursor and singly and doubly charged C-terminal y-ions are indicated. CE: collision energy used for fragmentation. Synthetic peptides were used to select diagnostic transitions and the optimal collision energies and to determine the retention time for the peptides of interest. For reference peptides Ref-1, Ref-2 and Ref-3 diagnostic transitions were selected from CID spectra.
pmic7205-sup-0016-FigureS3.tif1347KFigure S3: The kinases Prk1 and Hog1 phosphorylate Pan1. A: The phosphorylation status of the Prk1 consensus ([L/I/V/M]xx[Q/N/T/S]xTG) site T 570 of Pan1 remains unaltered in response to osmotic stress. SILAC labeled Pan1-HTBeaq was purified from wt, hog1Δ strain backgrounds. Untreated cultures were labeled with 13C arginine and 13C lysine; osmostressed (10 minutes 0.5 M NaCl) cultures with 12C arginine and 12C lysine. Phosphothreonine 570 has been previously identified as a target site of kinase Prk1 [12]. We obtained a 1:1 ratio for phosphopeptide T#GFGNNEIYTK in all experiments. B: In vitro kinase assay using Hog1-as and yeast purified TAP-tagged Pan1 as a substrate. Kinase reactions were done for 30 minutes in the presence (+) or absence (-) of 5μM of as-inhibitor SSP86.
pmic7205-sup-0001-si.doc141KSupplemental Material and Methods

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