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1 Supplementary Materials for In Vivo Phosphoproteomics Analysis Reveals the Cardiac Targets of β-adrenergic Receptor Signaling Alicia Lundby,* Martin N. Andersen, Annette B. Steffensen, Heiko Horn, Christian D. Kelstrup, Chiara Francavilla, Lars J. Jensen, Nicole Schmitt, Morten B. Thomsen, Jesper V. Olsen* *Corresponding author. (A.L.); (J.V.O.) Published 4 June 2013, Sci. Signal. 6, rs11 (2013) DOI: /scisignal This PDF file includes: Fig. S1. Electrophysiological effects of pharmacological βar modulation on murine hearts. Fig. S2. Evaluation and assessment of MS and MS/MS data quality. Fig. S3. Correlation plot analyses of biological replicates of phosphopeptide measurements. Fig. S4. Statistical analysis of identified peptides. Fig. S5. Western blotting analysis of Akt and GSK-3α in control and β 1 ARstimulated mice. Fig. S6. Localization of K V 7.1 channels and presence of βars in MDCK cells. Fig. S7. Trafficking of K V 7.1 Ser 27 and Ser 92 mutant channels in MDCK cells. Fig. S8. Testing of K V 7.1 wild-type, K V 7.1 S92A, and K V 7.1 S92D mutant channels coexpressed with KCNE1 in X. laevis oocytes. Legends for tables S1 to S5 Other Supplementary Material for this manuscript includes the following: (available at Table S1 (Microsoft Excel format). List of all identified modification-specific peptides. Table S2 (Microsoft Excel format). List of all identified class 1 phosphorylation sites. Table S3 (Microsoft Excel format). List of all identified proteins. Table S4 (Microsoft Excel format). List of all phosphorylation sites regulated by β 1 AR stimulation. Table S5 (Microsoft Excel format). List of all identified phosphorylated kinases.

2 Fig. S1. Electrophysiological effects of pharmacological βar modulation on murine hearts. (A to C) The time-course development of the cardiac RR interval duration, the time elapsing between two consecutive R waves, evaluated from electrocardiograms is shown for each of the three groups of mice. For each mouse, the RR interval was monitored for 5 min to ensure a stable heart rate before the experiment was begun. At each time point, the mean ± SEM RR interval duration is shown. At the time points indicated by arrows, either a βar modulator or saline infusion was started. *P < 0.05 for RR interval versus RR interval at t = 0 min (Student s t-test). (A) The control mice were infused with a cocktail of selective β 1 AR (metoprolol, 4.0 mg/kg IV) and β 2 AR (ICI , 2.5 mg/kg IV) antagonists at t = 0 min and saline at t = 10 min; n = 3 mice. (B) The β 1 AR-stimualted mice were infused with a selective β 2 AR antagonist (ICI , 2.5 mg/kg IV) at t = 0 min followed by a selective β 1 AR agonist (dobutamine, 1.5 mg/kg IV) at t = 10 min; n = 3 mice. (C) The β 2 AR-stimulated mice were infused with a selective β 1 AR antagonist (metoprolol, 4.0 mg/kg IV) at t = 0 min followed by a selective β 2 AR agonist (salbutamol, 2.5 mg/kg IV) at t = 10 min; n = 3 mice. RR intervals were comparable at baseline, and are reported every 5 min and at peak effect. Metroprolol, but not ICI , slowed heart rate, whereas dobutamine, and to a lesser degree salbutamol, increased heart rate. Right panels show exemplary surface ECG traces (lead I) at points indicated in the panels on the left panels (a, b, and c). In the surface ECGs, individual RR intervals are indicated between R waves.

3 Fig. S2. Evaluation and assessment of MS and MS/MS data quality. (A) Table with peptide statistics for phosphorylated and nonphosphorylated peptides. Unique peptides are the number of modification statespecific peptides with or without phosphorylation site(s). MS/MS scans are the total number of peptidespectrum matches in the dataset. Thus, each phosphopeptide was identified more than 30 times on average. (B) Peptide mass accuracy. The peptide intensities for all phosphopeptides are plotted as a function of calibrated peptide precursor mass errors measured for all identified peptides in parts-permillion (ppm). The absolute average mass accuracy is shown. (C) Fragment ion mass accuracy. HCD fragment ion errors were binned in 1-ppm units, and the distribution of counts per bin is shown. The absolute average mass accuracy was calculated. (D) Histogram illustrating the Mascot score distribution of all phosphopeptides. Mascot scores were binned in 10-score units, and each bin is displayed as a bar indicating the peptide count. The median and average Mascot scores are shown. (E) Histogram of Andromeda score distribution of all phosphopeptides. Andromeda scores were binned in 25-score units, and each bin is displayed as a bar indicating the peptide count. The median and average Andromeda scores are shown. (F) Distribution of phosphopeptide precursor intensities. Peptide intensities are binned in log 10 -magnitudes of log 10 -transformed, label-free, XIC-based intensities and are displayed by bars indicating the count in each bin. The counts are given to the right of each bar. (G) Spectral count distribution. Histogram of HCD-MS/MS spectra count for each identified phosphopeptide. The peptide count for each bin is given to the right of the corresponding bar.

4 Fig. S3. Correlation plot analyses of biological replicates of phosphopeptide measurements. For each mouse, the phosphopeptide intensities are plotted against the median phosphopeptide intensity for the group of mice to which the animal belongs. Correlation plots for measurements from mice in the β 1 ARstimualted group are shown in the top row, correlation plots for measurements from mice in the β 2 ARstimulated group are shown in the middle row, and correlation plots for measurements from mice in the control group are shown in the bottom row. The R 2 correlation coefficient is indicated in the corner of each plot. Measurements from the three technical replicate experiments that were performed for each mouse were averaged in the analyses presented. The technical replicate experiments were evaluated in a similar manner and their average R 2 correlation coefficients were (0.76, 0.84, and 0.76) for mice in the control group, (0.85, 0.79, and 0.76) for mice in the β 1 AR-stimulated group, and (0.73, 0.91, and 0.84) for mice in the β 2 AR-stimulated group.

5 Fig. S4. Statistical analysis of identified peptides. (A to C) The three volcano plots show negative logtransformed t-test derived P values (log 10 ) as a function of log-transformed ratios of average peptide intensities (log 2 ). All points represent a peptide, and the hyperbolic curves indicate permutation-based FDR cut-offs of In each plot, the number of peptides in the analysis, N, is indicated. (A) Analysis of peptide intensities for peptides without phosphorylation sites (unmodified) measured in β 1 AR-stimulated mice and compared to control mice. The permutation-based significance curve reveals that none of the peptides is statistically significantly different between the β 1 AR-stimulated mice and the control mice. (B) Analysis of unmodified peptide intensities measured in β 2 AR-stimulated mice and compared to control mice. None of the peptides is statistically significantly different between the β 2 AR-stimulated mice and the control mice. (C) Analysis of phosphopeptide intensities measured in β 2 AR-stimulated mice and compared to control mice. None of the peptides is statistically significantly different between the β 2 ARstimulated mice and the control mice.

6 Fig. S5. Western blotting analysis of Akt and GSK-3α in control and β 1 AR-stimulated mice. (A) Total heart lysates (100 µg) from control or β 1 AR-stimulated mice (three for each condition) were analyzed by Western blotting for pakt or pgsk-3a, and then for total Akt and total GSK-3A, as indicated. (B) Densitometric analysis of pakt and pgsk-3α normalized to their respective total proteins. *P < 0.05 (Student s t-test). Both kinases were phosphorylated to a larger extent in β 1 AR-stimulated mice than in control mice. Phosphorylation of Thr 308 activates Akt, whereas phosphorylation at Ser 21 inhibits GSK-3A. Hence, these experiments suggest that β 1 AR stimulation leads to the increased activity of Akt and the decreased activity of GSK-3α.

7 Fig. S6. Localization of K V 7.1 channels and presence of βars in MDCK cells. (A) The localization of K V 7.1 channels in MDCK cells can be controlled by a calcium switch (26) and evaluated by confocal microscopy. By performing such a calcium switch (see the Materials and Methods for details) we captured the channels when they were localized in the ER (top panel, 4 hours into the calcium switch) or at the basolateral part of the plasma membrane (middle panel, 27 hours into the calcium switch). When the K V 7.1 channels were at the plasma membrane, treating the cells with isoproterenol for 20 min did not affect their localization (bottom panel). K V 7.1 localization is visualized by red staining, actin is a marker for the cell surface and is visualized by green staining, and DAPI stains the nucleus and is visualized in blue. (B) The extent of expression of the genes encoding β 1 AR and β 2 AR in MDCK cells was tested based on previously published microarray data. Two data sets in which MDCK cells were profiled in two different biological settings were chosen. The processed data were downloaded from and An overall expression distribution is plotted for each data set. The colored vertical lines indicate the specific probe set intensity observed for each receptor in each experiment. The combined measurement for each receptor in each experiment is shown with a boxplot. Statistical tests were performed with a Wilcoxon rank sum test. For both experiments, expression of the gene encoding β 1 AR was statistically significantly higher than that of the gene encoding β 2 AR. (C) The extent of phosphorylation of K V 7.1 Ser 92 was compared in MDCK cells subjected to β 1 AR-specific stimulation or β 2 AR-specific stimulation relative to control cells treated with antagonists of β 1 AR and β 2 AR by label-free targeted LC-MS/MS. The median intensity ratio of the phosphopeptide VS(ph)IYSTR in each of the three experimental conditions relative to the control is visualized as boxplots. The extent of phosphorylation of K V 7.1 Ser 92 was statistically significantly higher in cells subjected to β 1 AR-specific stimulation than in both the control cells and the cells subjected to β 2 AR-specific stimulation (Student s t-test, P < 0.01).

8 Fig. S7. Trafficking of K V 7.1 Ser 27 and Ser 92 mutant channels in MDCK cells. (A and B) K V 7.1 residues Ser 27 and Ser 92 were mutated to either alanine (S27A, S92A) to abolish phosphorylation or aspartate (S27D, S92D) to mimic constitutive phosphorylation. Wild-type or mutant channels (green staining) were co-expressed in MDCK cells with ER-DsRed (red staining), which is a marker of the ER. Localization of the channels was evaluated by confocal microscopy. Representative images of all tested single mutants are shown in (A), whereas representative images of all tested double mutants are shown in (B). All mutant channels tested trafficked from the ER to the plasma membrane similarly to the wild-type channel.

9 Fig. S8. Testing of K V 7.1 wild-type, K V 7.1 S92A, and K V 7.1 S92D mutant channels coexpressed with KCNE1 in X. laevis oocytes. (A) Representative current traces for K V 7.1/KCNE1 and K V 7.1 S92A/KCNE1 (left) or for K V 7.1/KCNE1 and K V 7.1 S92D/KCNE1 (right) eliciting I Ks -like currents. The current traces before (black) and after (gray) incubation with 8-bromo-cAMP are shown. All electrophysiological recordings were made by applying the depicted voltage-step protocol; 2-s duration voltage steps in 20-mV increments to +60 mv from a holding potential of -80 mv followed by a step to mv. (B) Proteins were extracted from X. laevis oocytes co-expressing either K V 7.1 and KCNE1 or K V 7.1 S92A and KCNE1. Channel protein abundance was quantified based on Western blots that were normalized relative to actin protein abundance (n = 3 experiments). There was no statistically significant difference in the abundance of wild-type and mutant channels (student s t-test). (C) Electrophysiological recordings from oocytes co-expressing K V 7.1 S92D and KCNE1. The current amplitudes were measured before and after stimulation with camp, and are shown as means ± SEM (n = 25 oocytes from 3 batches of oocytes). (D) The fold-change in current amplitude induced by camp measured at 60mV. The foldchange in current amplitude after stimulation with camp was statistically significantly different for the mutant channels compared to wild-type channel. *P < 0.05 (Student s t-test).

10 Table S1. List of all identified modification-specific peptides. The first sheet contains all of the phosphorylated peptides and the second sheet contains all of the other peptides, that is, peptides that were not phosphorylated. The third sheet contains explanations for the column headers. Table S2. List of all identified class 1 phosphorylation sites. The first sheet lists all of the sites, whereas the second sheet contains explanations for the column headers. Table S3. List of all identified proteins. The first sheet lists all of the identified proteins, whereas the second sheet contains explanations for the column headers. Table S4. List of all phosphorylation sites regulated by β 1 AR stimulation. The first sheet lists all of the phosphorylation sites regulated in response to β 1 AR stimulation. Sites residing on phosphopeptides with increased intensity in the β 1 AR mice compared to the control mice are indicated by an up and those residing on phosphopeptides with decreased intensity are indicated by a down in the column labeled Increased or decreased in abundance?. The second sheet contains explanations for the column headers. Table S5. List of all identified phosphorylated kinases. The first sheet lists all of the kinases. The gene and protein names for each kinase and its IPI and Uniprot identifiers are listed together with the amino acid sequences of the identified phosphorylated peptides. Peptides that were statistically significantly regulated upon β 1 AR stimulation are indicated. The second sheet contains an explanation of the column headers.