Quantitative phosphoproteomics of vasopressin-sensitive renal cells: regulation of aquaporin-2 phosphorylation at two sites

Abstract

Protein phosphorylation plays a key role in vasopressin signaling in the renal-collecting duct. Large-scale identification and quantification of phosphorylation events triggered by vasopressin is desirable to gain a comprehensive systems-level understanding of this process. We carried out phosphoproteomic analysis of rat inner medullary collecting duct cells by using a combination of phosphopeptide enrichment by immobilized metal affinity chromatography and phosphorylation site identification by liquid chromatography-mass spectrometry(n) neutral loss scanning. A total of 714 phosphorylation sites on 223 unique phosphoproteins were identified from inner medullary collecting duct samples treated short-term with either calyculin A or vasopressin. A number of proteins involved in cytoskeletal reorganization, vesicle trafficking, and transcriptional regulation were identified. Previously unidentified phosphorylation sites were found for membrane proteins essential to collecting duct physiology, including eight sites among aquaporin-2 (AQP2), aquaporin-4, and urea transporter isoforms A1 and A3. Through label-free quantification of phosphopeptides, we identified a number of proteins that significantly changed phosphorylation state in response to short-term vasopressin treatment: AQP2, Bclaf1, LRRC47, Rgl3, and SAFB2. In the presence of vasopressin, AQP2 monophosphorylated at S256 and diphosphorylated AQP2 (pS256/261) increased in abundance, whereas AQP2 monophosphorylated at S261 decreased, raising the possibility that both sites are involved in vasopressin-dependent AQP2 trafficking. This study reveals the practicality of liquid chromatography-mass spectrometry(n) neutral loss scanning for large-scale identification and quantification of protein phosphorylation in the analysis of cell signaling in a native mammalian system.

Fig. 1.

Experimental approach. (A) Rat IMCD protein samples were digested with trypsin, followed by separation by strong cation exchange (SCX) chromatography. Phosphorylated peptides were enriched from total peptides by using IMAC. (B) Phosphopeptide samples were analyzed by LC-MSⁿ neutral loss scanning in which fragmentation of the phosphopeptide resulted in loss of phosphoric acid (−98 Da; neutral loss peak) from the full MS (parent ion) scan. This neutral loss peak was selected for further fragmentation and peptide identification in the MS³ spectrum.

Fig. 2.

Identification and confirmation of phosphorylation sites using LC-MSⁿ neutral loss scanning. (A) Confirmation of AQP2 phosphorylation on S256 (∗) from an MS³ spectrum (Left). Arrows indicate specific peaks in the mass spectrum that aided in distinguishing the actual site of phosphorylation from other potential sites. Confirmation of phosphorylation on S256 as well as identification of a new site, S261, for a doubly phosphorylated AQP2 peptide from an MS⁴ spectrum (Right). (B) Comparison of different MS levels for Rho GTPase activating protein 24, a phosphoprotein that was identified solely on the quality of its MS⁴ spectrum. In both A and B, asterisks indicate residues that were phosphorylated in the full MS scan but subsequently underwent neutral loss leaving a modified original residue mass without water (−18 Da).

Fig. 3.

Quantification of AQP2 phosphorylation in response to short-term dDAVP treatment by immunoblotting. (A) IMCD suspensions treated with dDAVP (10⁻⁹ M) or without (control) for 10 min. Immunoblots were probed by using a phosphospecific AQP2 antibody that recognizes phosphorylated S256. (B) Phosphorylated AQP2 levels were significantly increased with dDAVP treatment (dDAVP 237 ± 31.4% vs. control 100 ± 21.5%; ∗, P < 0.05).

Fig. 4.

MS quantification of AQP2 phosphorylation in response to dDAVP treatment. (A) Aligned chromatogram plots of overall peptide abundance for AQP2 phosphorylated at both S256 and S261. Retention time in minutes (x axis) and intensity (y axes). The doubly phosphorylated AQP2 peptide increased an average of 2.67-fold with dDAVP treatment. Values were calculated by using the “area under the curve” between the two cutoff lines (dashed lines). (B) Targeted ion selection (TIS) analysis of singly phosphorylated AQP2 peptides. Labeled peaks corresponding to either pS261 (▾) or pS256 (▿) indicate spectra that were confirmed by manual validation. Max, maximum peak intensity.