Abstract
We have performed LC‐MS/MS analysis of differential centrifugation fractions from rat IMCD to evaluate the utility of differential centrifugation in assessing trafficking of the water channel aquaporin‐2 (AQP2). Freshly prepared IMCD suspensions were homogenized and subjected to sequential centrifugations at 1K g, 4K g, 17K g, and 200K g. Preliminary immunoblotting confirmed that the ratio of AQP2 in the 17K fraction to the 200K fraction underwent an increase in response to the vasopressin analog dDAVP, largely due to a reduction in the 200K fraction. Immunoblotting revealed that the major phosphorylated forms of AQP2 were present in both the 17K and 200K fractions. LC‐MS/MS analysis showed that markers of 'intracellular vesicles' were present in both the 17K and 200K fractions. In contrast, plasma membrane proteins were predominantly present in the 4K and 17K fractions. Proteins associated with several multiprotein complexes (e.g. proteasomes and actin‐related protein 2/3 complex) were virtually exclusively present in the 200K fraction. Overall, we identified 656 proteins, including 189 not previously present in the IMCD Proteome Database. The data show that both the 17K and 200K fractions are highly heterogeneous and cannot be equated with 'plasma‐membrane' and 'intracellular vesicle' fractions, respectively. Alternative methods for assessment of AQP2 trafficking are recommended.
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