Supplemental materials for “Quantitative Proteomics Identifies Vasopressin- Responsive Nuclear Proteins in the Collecting Duct” Schenk et al. Page 2. Supplemental Fig. 1. Page 3. Supplemental Methods Page 7. Supplemental Table 1 Page 87. Supplemental Table 2 Page 212. Supplemental Table 3 Page 219. Supplemental Table 4 Page 221. Supplemental Table 5 Page 222. Supplemental Table 6 Page 315. Supplemental Table 7 Page 317. Supplemental Table 8 Incorporation of labeled amino acids (9 days, >6 doubling times) Light medium Heavy medium [12C] Lysine [13C] Lysine [12C,14N] Arginine [13C,15N] Arginine Growth to confluency on membrane support (5 days, in presence of 0.1nM dDAVP) Withdrawal of dDAVP (24 h) Stimulation with dDAVP or vehicle (60 min) Pooling of cells 1:1 from each populations ; ,; , ... :. ;; ,.. ; .. :. ; .. ; , ;. ,;: ..;.. , . ; ,. : ; , Nucleo- cytoplasmic separation (NE-PER®) Cytoplasmic extract Nuclear extract Nuclear pellet In-gel trypsin digestion, fractionation LC-MS/MS (Orbitrap) Protein identification and quantitation Full Methods Cell culture and sample preparation. All studies were done in an AQP2-expressing mpkCCD clonal cell line (clone 11)1 grown on membrane supports (Transwell, Corning). We carried out three series of mass spectrometry experiments, viz. a non-quantitative profiling study and two series of experiments using the SILAC method for quantification. In each study, stepwise separation of cytoplasmic and nuclear extract was performed using a commercially- available kit (NE-PER® Nuclear and Cytoplasmic Extraction Reagent Kit, Pierce, Rockfort, IL). In the non-quantitative profiling experiments, the test protocol supplied by the manufacturer was followed to obtain cytoplasmic extract, nuclear extract and nuclear pellet. To enhance the purity of the separation of cytoplasm and nucleus for the quantitative studies, the steps extracting the cytoplasm were carried out twice before proceeding with the nuclear sample. The two series of studies quantifying the response to the vasopressin analog dDAVP (0.1 nM) were done using SILAC14 culture media containing the amino acids arginine and lysine 13 15 13 12 14 labeled with stable isotopes (“heavy”: C6 N4 arginine, C6 lysine; “light”: C6 N4 arginine, 12 C6 lysine [Invitrogen]) for 16 days (three passages). This labeling period is sufficient to achieve >98% saturation of labeling.2 At the beginning of an experiment (Suppl. Fig. 1), labeled cells were grown in the presence of dDAVP (0.1 nM) for 4-5 days to assure that all vasopressin- dependent proteins are expressed.3 In the first quantitative series, the dDAVP was then withdrawn for 24 hour prior to re-exposure to vasopressin or vehicle for 60 minutes. In the second series, observations were made after 30-min exposure to dDAVP after a 6-hour washout period. For these experiments, cells (60-90 µg protein) were pooled in a 1:1 ratio (dDAVP:vehicle) and subjected to nuclear and cytoplasmic separation as described above prior to mass spectrometry. Mass spectrometry. Samples were reduced with DTT (10 mM), alkylated with iodoacetamide (40 mM), and solubilized with ¼ part of Laemmli buffer (5×: 7.5% SDS, 30% glycerol, 50 mm Tris, pH 6.8) prior to 1D-SDS-PAGE (4-15% gradient). One-dimensional SDS- PAGE was performed using 4%–15% gradient polyacrylamide gels. Protein in the gel was visualized with colloidal Coomassie blue staining (GelCode Blue Stain Reagent, G-250; Pierce Biotechnology, Rockford, IL) for 5 min followed by destaining in deionized H2O for 1 hour. Protein in the gel was then sliced into small blocks for a total of 20 or 40 blocks per lane. Each block was minced into small pieces (1–1.5 mm3). Gel pieces from each block were further destained and dehydrated by incubating with 25 mm NH4HCO3/50% acetonitrile (ACN) solution for 10 min three times and then were dried in vacuo. The dried gel pieces were immersed in 25 mm NH4HCO3 solution containing 12.5 ng/μl trypsin (V5113) (Promega Corp., Madison, WI) and incubated at 37°C overnight. The tryptic peptides were extracted by incubating the gel pieces with 50% ACN/0.1% formic acid (FA) followed by sonication in a water bath for 20 min. This step was repeated twice. The volume of the extracted peptide samples was reduced to about 5 μl in vacuo and then the samples were reconstituted to 20 μl in 0.1% FA. The resulting peptide mixtures were concentrated and desalted with C18 Zip-tips (Millipore Corp., Bedford, MA). They were dried in vacuo and reconstituted to 10 μl in 0.1% FA for LC-MS/MS analysis. Tryptic peptides were analyzed on an Eksigent nanoLC system connected to an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) equipped with a nano-electrospray ion source. Peptides were loaded onto a peptide trap cartridge (Agilent Technologies) at a flow rate of 6 μl/min. The trapped peptides were then fractionated with a reversed-phase PicoFrit column (New Objective, Woburn, MA) using a linear gradient of 5%–35% ACN in 0.1% FA. The gradient time was 45 min at a flow rate of 0.25 μl/min. Precursor mass spectra (MS1) were acquired in the orbitrap at 60,000 resolution and product mass spectra (MS2) were acquired with the ion trap. MS spectra were searched using the SEQUEST, Inspect, and OMSSA algorithms as described4 using the most recent mouse RefSeq Database. Data were filtered using the target- decoy approach5 to obtain <1% FDR at a peptide level. Quantification of light and heavy peptides was performed by QUIL software.6 Uniqueness or multiplicity of putative peptide assignment was evaluated using ProMatch software4. Protein quantifications were median- normalized in each of experimental pair. Dual statistical criteria were applied: p, t-test (paired, P<0.05) and outside the 95% confidence interval defined by vehicle vs. vehicle experiments. In an additional set of SILAC experiments, we analyzed samples treated for 30 min with dDAVP (0.1 nM) rather than 60 min (see above). Protein samples were reduced with 10 nM DTT for 1 hour and alkylated with iodoacetamide (40 nM) for 1 hour in the dark. Samples were o diluted in 25 mM NH4HCO3 before addition of trypsin (1:20 wt/wt for 16 hour at 37 C). After acidifying and desalting (HLD cartridges; Oasis), samples were dried in vacuo and fractionated using strong cation exchange (SCX) chromatography. Samples were resuspended in solvent A (5 mM KH2PO4, 25% ACN, pH 3.0) and injected onto a Polysulfoethyl A SCX column (4.6 mm ID x 20 cm length, 5 um particle size and 300 A pore size; PolyLC) on an Agilent HP1100 system. A total of 24 fractionated samples for the NE and 24 fractionated samples for NP were obtained. The fractionated samples were dried in vacuo and resuspended in 0.1% formic acid. Flow-through samples from Fe-NTA phosphopeptide IMAC columns (Pierce) were analyzed by LC-MS/MS after desalting using Graphite Spin columns (Pierce). Bioinformatics. Clustering of transcription factors was done using the Muscle algorithm7 and results were displayed as a dendrogram using iTOL (Interactive Tree Of Life).8 Conserved transcription factor binding sites (TFBS) in the 5’-flanking regions of specific genes were found using the online Genomatix software suite and TFBS database (http://www.genomatix.de/). Immunoblotting and antibodies. Immunoblot analysis using nitrocellulose membranes and IR dye-coupled secondary antibodies was performed as described previously9. Blocking was performed using commercially-available blocking buffer (Licor). Fluorescence signals were read out using the Odyssey system. Reference List 1. Yu MJ, Miller RL, Uawithya P, Rinschen MM, Khositseth S, Braucht DW, Chou CL, Pisitkun T, Nelson RD, Knepper MA: Systems‐level analysis of cell‐specific AQP2 gene expression in renal collecting duct. Proc Natl Acad Sci U S A 106:2441‐2446, 2009 2. Rinschen MM, Yu MJ, Wang G, Boja ES, Hoffert JD, Pisitkun T, Knepper MA: Quantitative phosphoproteomic analysis reveals vasopressin V2‐receptor‐dependent signaling pathways in renal collecting duct cells. Proc Natl Acad Sci U S A 2010 3. Khositseth S, Pisitkun T, Slentz DH, Wang G, Hoffert JD, Knepper MA, Yu MJ: Quantitative protein and mRNA profiling shows selective post‐transcriptional control of protein expression by vasopressin in kidney cells. Mol Cell Proteomics 2010 4. Tchapyjnikov D, Li Y, Pisitkun T, Hoffert JD, Yu MJ, Knepper MA: Proteomic profiling of nuclei from native renal inner medullary collecting duct cells using LC‐MS/MS. Physiol Genomics 40:167‐183, 2010 5. Elias JE, Gygi SP: Target‐decoy search strategy for increased confidence in large‐scale protein identifications by mass spectrometry 2. Nat Methods 4:207‐214, 2007 6. Wang G, Wu WW, Pisitkun T, Hoffert JD, Knepper MA, Shen RF: Automated quantification tool for high‐throughput proteomics using stable isotope labeling and LC‐MSn. Anal Chem 78:5752‐5761, 2006 7. Edgar RC: MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5:113, 2004 8. Letunic I, Bork P: Interactive Tree Of Life v2: online annotation and display of phylogenetic trees made easy. Nucleic Acids Res 39:W475‐W478, 2011 9. Pisitkun T, Jacob V, Schleicher SM, Chou CL, Yu MJ, Knepper MA: Akt and ERK1/2 pathways are components of the vasopressin signaling network in rat native IMCD. Am J Physiol Renal Physiol 295:F1030‐F1043, 2008 Suppl. Tab. 1 List of identified proteins; excludes proteins without mRNA match; peptide count for the three compartments is given in columns D-F GI Accession Cytoplasmic Nuclear Nuclear No. Gene Symbol Protein Name extract extract pellet 27228985 Ndufa12