BASIC RESEARCH www.jasn.org Phosphoproteomic Profiling Reveals Vasopressin- Regulated Phosphorylation Sites in Collecting Duct Amar D. Bansal,* Jason D. Hoffert,* Trairak Pisitkun,* Shelly Hwang,* Chung-Lin Chou,* Emily S. Boja,† Guanghui Wang,† and Mark A. Knepper* *Epithelial Systems Biology Laboratory, and †Proteomics Core Facility, Division of Intramural Research, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland ABSTRACT Protein phosphorylation is an important component of vasopressin signaling in the renal collecting duct, but the database of known phosphoproteins is incomplete. We used tandem mass spectrometry to identify vasopressin-regulated phosphorylation events in isolated rat inner medullary collecting duct (IMCD) suspensions. Using multiple search algorithms to identify the phosphopeptides from spectral data, we expanded the size of the existing collecting duct phosphoproteome database from 367 to 1187 entries. Label-free quantification in vasopressin- and vehicle-treated samples detected a significant change in the phosphorylation of 29 of 530 quantified phosphopeptides. The targets include important structural, regulatory, and transporter proteins. The vasopressin-regulated sites included two known sites (Ser-486 and Ser-499) present in the urea channel UT-A1 and one previously unknown site (Ser-84) on vasopressin-sensitive urea channels UT-A1 and UT-A3. In vitro assays using synthetic peptides showed that purified protein kinase A (PKA) could phosphorylate all three sites, and immunoblotting confirmed the PKA dependence of Ser-84 and Ser-486 phosphorylation. These results expand the known list of collecting duct phosphoproteins and highlight the utility of targeted phosphoproteomic approaches. J Am Soc Nephrol 21: 303–315, 2010. doi: 10.1681/ASN.2009070728 Vasopressin plays a central role in collecting duct In a previous study,17 we used tandem mass physiology. Signaling through the V2 receptor re- spectrometry (LC-MS/MS)-based quantitative sults in an increase of cAMP levels and causes acti- phosphoproteomics to partially annotate the phos- vation of protein kinase A (PKA).1,2 In addition, phoproteome of rat inner medullary collecting duct over 200 other serine/threonine protein kinases are (IMCD). We subsequently quantified the differen- expressed in native collecting duct cells,3 and some tial phosphorylation of four serine residues (Ser- of these have been shown to play important roles in 256, Ser-261, Ser-264, and Ser-269) in the C-termi- the response to vasopressin.4–10 Vasopressin signal- nal tail of rat aquaporin-2 (AQP2) in response to ing is important not only for regulation of water short-term exposure to the vasopressin analog transport through aquaporins11 but also for regula- dDAVP.17,18 We also found a number of phosphor- tion of urea12 and sodium transport.13,14 Vasopres- sin also regulates long-term gene expression of col- Received July 15, 2009. Accepted November 2, 2009. lecting duct proteins, such as aquaporins.15,16 Because protein phosphorylation plays a central Published online ahead of print. Publication date available at www.jasn.org. role in vasopressin signaling, the identification and quantification of phosphorylated proteins in re- Correspondence: Dr. Mark A. Knepper, National Institutes of Health, 10 Center Drive, Building 10, Room 6N260, Bethesda, sponse to vasopressin are essential to understand- MD 20892-1603. Phone: 301-496-3064; Fax: 301-402-1443; E-mail: ing the mechanism of action of this hormone in [email protected] collecting duct. Copyright ᮊ 2010 by the American Society of Nephrology J Am Soc Nephrol 21: 303–315, 2010 ISSN : 1046-6673/2102-303 303 BASIC RESEARCH www.jasn.org A BC IMCD isolate IMCD isolate IMCD isolate dDAVP treatment dDAVP treatment control dDAVP treatment control Protein isolation Protein isolation Protein isolation Protein isolation Protein isolation Proteolysis Proteolysis Proteolysis Proteolysis Proteolysis SCX fractionation IMAC IMAC IMAC IMAC IMAC LC-MS/MS Profiling large-scale quantification large-scale quantification TIS quantification TIS quantification Figure 1. The three different LC-MS/MS experiments performed are as follows: (A) The initial phosphoproteomic profiling experiment consisted of a single dDAVP-treated sample that was processed using SCX fractionation. (B) The second experiment was performed for large-scale quantification using a nonselective “profiling” mode. (C) The third experiment was performed for targeted quantification by selection of precursor ion m/z ratios, so-called targeted ion selection (TIS) mode. ylation sites on the vasopressin-sensitive urea channel, UT- dDAVP-treated IMCD sample (Figure 1A) as described in A.17 However, because of the limited sensitivity of the experi- “Concise Methods,” followed by phosphopeptide enrichment mental approach, we were unable to quantify changes in via immobilized metal affinity chromatography (IMAC). Sam- phosphorylation at these sites in UT-A, despite evidence for ples representing 24 SCX fractions were analyzed on a Thermo such sites on the basis of previous studies.19,20 LTQ mass spectrometer, and the resulting spectra were One of the primary aims of this study was to increase the searched using three different search algorithms: SEQUEST, sensitivity of our MS-based workflow to annotate a larger por- InsPecT, and OMSSA. Figure 2 shows a Venn diagram of tion of the IMCD phosphoproteome. This was accomplished unique phosphopeptides identified from each of the three in three ways: (1) by implementing an effective, chromatogra- search algorithms. All datasets were filtered for a Ͻ2% false- phy-based stratification technique for our peptide samples, (2) discovery rate on the basis of target-decoy analysis.21 Overlap- by using a higher resolution mass spectrometer, and (3)by using multiple proteomic search algorithms to process the MS OMSSA InsPecT data. Using these combined approaches, we increased coverage 1322 2120 of the IMCD phosphoproteome by approximately 3-fold com- pared with the previous study.17 In addition, we present large- scale phosphoproteomic data quantifying the effect of vaso- 206 pressin on phosphorylation of IMCD proteins. Last, we 279 identify and quantify six phosphorylation sites on the vaso- pressin-sensitive urea channel (isoforms A1 and A3) and dem- 948 735 onstrate that three of these sites undergo large increases in 102 phosphorylation in response to short-term dDAVP treatment. 231 206 RESULTS SEQUEST Phosphoproteomic Profiling of IMCD 1274 Our previous study using an LC-MS/MS “shotgun” approach identified 223 unique phosphoproteins in native IMCD cells.17 One of the aims of this study was to expand the size of the Combined total 2707 identified collecting duct phosphoproteome by adding meth- Figure 2. Venn diagram shows the number of unique phos- ods that would increase overall sensitivity; viz., using sample phopeptide identifications that resulted from each of three search fractionation via strong cation exchange (SCX) and employing algorithms (SEQUEST, InsPecT, and OMSSA). A false-discovery multiple MS search algorithms. SCX was performed on a single rate stringency of Ͻ2% was used for each of the searches. 304 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 303–315, 2010 www.jasn.org BASIC RESEARCH kDa control dDAVP (10-9 M) mentation (Figure 1B). All 10 samples were searched with SE- QUEST (false-discovery rate Ͻ2%), and relative quantifica- 37 AQP2 22 Ser(p)-256 tion was performed using QUOIL. A total of 530 unique 25 phosphopeptides from 278 proteins were quantified (Figure 4). For statistical analysis, values were converted to log2 of the Figure 3. Immunoblot showing that dDAVP increased phosphor- ratio (D/C), where D and C refer to the normalized areas of the ylation of Ser-256 of AQP2. An immunoblot of IMCD protein extracted peptide ion chromatograms under dDAVP-treated isolate shows five pairs of control and dDAVP-treated samples. and control conditions, respectively. Brackets in Figure 4 indi- Bands at 29 and 37 kDa indicate Ser(p)-256 bands (nonglycosy- cate the subset of 54 phosphopeptides with log (D/C) values lated and glycosylated forms, respectively). 2 either greater than 0.58 or less than Ϫ0.58. Of these 54 phos- ping regions consist of phosphopeptides that were identified phopeptides, 29 represented phosphopeptides with statisti- by more than one search algorithm and indicate a match not cally significant (P Ͻ 0.05) changes in abundance between the only of the phosphopeptide primary amino acid sequence but five paired control and dDAVP-treated IMCD tubules. These also for the exact site(s) of phosphorylation. A total of 735 phosphopeptides are shown in Table 1 and are marked with a phosphopeptides were identified by all three search algorithms superscript letter a. Phosphopeptides whose abundance did (as shown by the central overlap region). InsPecT identified not change significantly are marked with a superscript letter c the largest number of unique phosphopeptides (2120), with and were included as an internal negative control showing that the highest number of identifications that were not shared with the changes in phosphopeptide abundances found are most either of the other two programs (948). Annotated phos- likely not due to changes in the amount of protein analyzed. phopeptide data from all three searches are accessible online at Supplementary Table 1 is a complete list of phosphopeptides Ϫ the Collecting Duct Phosphoprotein Database (http://dir. with mean