Proteomics and AQP2 regulation

Abstract

The advent of modern quantitative protein mass spectrometry techniques around the turn of the 21st century has contributed to a revolution in biology referred to as 'systems biology'. These methods allow identification and quantification of thousands of proteins in a biological specimen, as well as detection and quantification of post-translational protein modifications including phosphorylation. Here, we discuss these methodologies and show how they can be applied to understand the effects of the peptide hormone vasopressin to regulate the molecular water channel aquaporin-2. The emerging picture provides a detailed framework for understanding the molecular mechanisms involved in water balance disorders.

Keywords: AQP2; aquaporin‐2; collecting duct; kidney; next‐generation DNA sequencing; phosphoproteomics; protein kinase A; protein mass spectrometry; proteomics; vasopressin.

Figure 1.. SILAC quantification of protein abundance changes in response to vasopressin analog dDAVP.

(A) Cultured cells are grown and equilibrated in medium containing either arginine and lysine with the normal abundance C and N isotopes, i.e. C-12 and N-14 (‘light medium’) or with arginine and lysine containing C-13 and N-15 (‘heavy medium’). Samples are trypsinized, mixed 1:1 and subjected to LC-MS/MS-based quantification. Individual tryptic peptides are ‘seen’ twice by the mass spectrometer at different molecular masses. The relative height of the light and heavy peaks reports the relative abundance of the parent protein in the original samples. (B) ‘Volcano plot’ reports the proteome-wide response to dDAVP versus vehicle, showing n=786 quantified proteins. Vertical axis is the negative of the base 10 logarithm of calculated P values (from t-statistic) for multiple replicates. Horizontal axis the base 2 logarithm of the abundance ratio dDAVP/Vehicle. Note the marked increase in abundance of AQP2 protein, which exceeds that of all other proteins.

Figure 2.. Dynamic SILAC-based global determination of protein half-lives and translation rates.

(A) Cultured cells are grown and equilibrated in medium containing arginine and lysine with the normal abundance C and N isotopes, i.e., C-12 and N-14 (‘light medium’). At time zero, cells are switched to ‘heavy medium’ with arginine and lysine containing C-13 and N-15. Thus, individual tryptic peptides are ‘seen’ twice by the mass spectrometer at different molecular masses indicating peptide synthesized before and after the medium switch. Sampling at different times allows assessment of the rate of protein turnover. (B) An example of data from the method quantifying a tryptic peptide corresponding to the small GTP-binding protein Rab11b (amino acid sequence: NNLSFIETSALDSTNVEEAFK). Data are fit to single-exponential curves with high precision. Both degradation rate (red) and production rate (blue) can be calculated from the data, which are equal if steady state conditions are maintained. Typically, several tryptic peptides derived from a single protein can be quantified, giving a very high degree of overall precision. (C) Volcano plot showing whole proteome quantification of effect of vasopressin analog dDAVP on translation rates of individual proteins. Labeled proteins have been identified in prior studies as having roles in vasopressin signaling or AQP2 regulation. Note that dDAVP exposure results in a multi-fold increase in AQP2 translation far exceeding changes in all other proteins.

Figure 3.. Phosphoproteomics.

(A) TMT for quantification in protein mass spectrometry. (B) Sequence logo based on the 33 phosphorylation sites that are increased by vasopressin in both mpkCCD cells and native IMCD cells (Table 1). (C) Sequence logo based on the 18 phosphorylation sites that are decreased by vasopressin in both mpkCCD cells and native IMCD cells (Table 2).