Comprehensive Proteomics in Vasopressin‐Sensitive mpkCCD Cells: Virtual Western Blotting

Vasopressin regulates the water channel aquaporin‐2 in collecting duct cells. Identification of the mechanisms involved depends on knowledge of the genes expressed. To obtain a comprehensive proteome list for vasopressin‐sensitive mpkCCD cells, we used protein mass spectrometry to identify proteins after two levels of fractionation: differential centrifugation followed by SDS‐PAGE. After slicing the resulting gels into 43 consecutive blocks, the gel blocks were subjected to in‐gel trypsinization followed by LC‐MS/MS. After filtering out poor spectra (target‐decoy analysis), 6766 unique proteins were identified. All peptides for each protein were mapped back to the gel‐block of origin to create a 'virtual western blot' for each protein. A histogram of apparent/calculated MW (ρ) for all proteins in the 17,000 Xg fraction revealed a mode of 1.05 and a median of 1.08, indicating that most proteins run at an apparent MW consistent with values calculated by summing the residue masses for all amino acids. However, a moderate fraction of proteins had either a high ρ (>1.45, n=475) or low ρ (<0.65, n=309). Bioinformatic analysis of the high ρ group indicated that a large fraction of these proteins are annotated as “N‐linked glycosylated” or “DNA binding”. The latter have positively‐charged DNA‐binding regions. The low ρ group had significantly lower pI values and shorter half‐lives than the total population. Many of the proteins in the low ρ group are known to undergo physiological cleavage (e.g. presenilin‐1, syndecan‐1) or are resident lysosomal proteins. Interestingly, aquaporin‐2 showed an aberrant band at ~17 kD (detected both by MS and western blotting) that could be a cleaved or alternatively spliced isoform of aquaporin‐2. The data provide a resource for future studies of vasopressin action.