Interestingly, we also observed extensive protein carbamylation in cortex and outer medulla, regions that have much lower physiological levels of urea. This finding is consistent with prior observations that proteins expressed in other organs with low local urea concentrations such as aquaporin, aBcrystallin, and TIMP-2 can undergo physiological carbamylation in the absence of elevated urea concentrations. Various proteins have also been shown to undergo increases in carbamylation in association with uremic levels of circulating urea. The mass spectrometry analysis of rat inner Kaempferide medulla identified 456 unique carbamylation sites in 403 proteins. Figure 4 shows the distribution of these carbamylation sites by their locations in the proteins. The largest group consists of sites that occur at the N-terminal amino acid of the proteins. In addition, a significant number of peptides showed carbamylation of N-termini that are nominally in internal regions of the protein. It is unlikely that these sites are established at the time of trypsinization of the samples because of the exhaustive procedures used to deplete the samples of urea. Indeed, 56 of 127 of these N-terminal sites were carbamylated at nontrypsin sites, i.e., at sites not preceded by lysine or arginine. We propose that these internal sites are present in the intact animal as a result of the action of physiological Gentiopicrin proteolytic processes, including the protein degradation that occurs as part of normal turnover of proteins in the cell. Note that many proteases in addition to trypsin, e.g. furin, cut after basic amino acids. Therefore, the remaining 71 sites may be targeted by endogenous members of this trypsin-like protease family. The results are summarized in Figure 6. Overall, slightly more than seven percent of identified proteins were found to be carbamylated and all compartments are represented. We speculate that the lower fraction of lysosomal proteins that are carbamylated may be because of the low pH inside of lysosomes, which would titrate the cyanate ion to cyanic acid. This could alter the protein folding or its ability to interact with other macromolecules. Another possibility is that carbamylation of lysines, arginines, cysteines, and protein N-termini may prevent other post-translational modifications. For example, lysines undergo multiple post-translational modifications including sumoylation, ubiquitylation, and acetylation. These can have important consequences with regard to the function and stability of the target protein. For example, ubiquitylation is known to mark proteins for degradation either by the proteasome pathway or via lysosomes. In general the molecular weight inferred from the mass spectrometry studies agrees with the nominal molecular weight of the individual proteins. We conclude from these observations that protein carbamylation occurs endogenously in the kidney and is present in multiple proteins that play important physiological roles. However, contrary to the hypothesis expressed in the Introduction, protein carbamylation does not appear to be restricted to the inner medulla where urea is highest in the kidney. Nor is there a striking difference in carbamylation in the inner medulla as a result of variation in water intake, which is associated with striking changes in levels of tissue urea in the renal inner medulla. It seems likely that circulating levels of urea, typically 4 mM in nondiuretic rats, is sufficient to result in carbamylation, providing an explanation for previous findings that several nonrenal proteins can be carbamylated. Currently approximately 1,400 species placed into 29 formal subgenera and 39 informal species groups are recognized worldwide.