LC-MS/MS analysis of differential centrifugation fractions from native inner medullary collecting duct of rat

We carried out LC-MS/MS based proteomic profiling of differential centrifugation fractions from rat inner medullary collecting duct (IMCD): 1) to provide baseline knowledge of the IMCD proteome and 2) to evaluate the utility of differential centrifugation in assessing trafficking of the water channel aquaporin-2 (AQP2). IMCD suspensions were freshly prepared from rat kidneys using standard methods. Homogenized samples were subjected to sequential centrifugations at 1,000, 4,000, 17,000, and 200,000 g. These samples, as well as the final supernatant, were subjected to LC-MS/MS analysis. Preliminary immunoblotting confirmed that the ratio of AQP2 in the 17,000-g fraction to the 200,000-g fraction underwent an increase in response to the vasopressin analog dDAVP, largely due to a reduction in the 200,000-g fraction. Immunoblotting for the major phosphorylated forms of AQP2 revealed that phosphorylated AQP2 was present in both the 17,000- and 200,000- g fractions. LC-MS/MS analysis showed that markers of "intracellular vesicles," chiefly endosomal markers, were present in both the 17,000- and the 200,000- g fractions. In contrast, plasma membrane proteins were predominantly present in the 4,000- and 17,000- g fractions. Proteins associated with several multiprotein complexes (e. g., actin-related protein 2/3 complex and proteasome complex) were virtually exclusively present in the 200,000- g fraction. Overall, we identified 656 proteins, including 189 not previously present in the IMCD database. The data show that both the 17,000- and 200,000- g fractions are highly heterogeneous and cannot be equated with " plasma membrane" and "intracellular vesicle" fractions, respectively, leading us to propose an alternative approach for use of differential centrifugation to assess vesicular trafficking to the plasma membrane.