Protein carbamylation is a physiological post‐translational modification in the renal inner medulla

Abstract

Listen

Translate article

We hypothesize that the high urea concentration in the renal inner medulla (IM) is associated with physiological carbamylation of proteins. Using a carbamylation‐specific antibody, immunoblotting revealed multiple bands in the kidney of antidiuretic rats with a gradient of carbamylation from cortex to IM, suggestive of large‐scale protein carbamylation. To test the specificity of the antibody, we incubated BSA with 1 M urea in PBS or PBS alone for 4 days. The antibody recognized a band consistent with carbamylated BSA in the presence but not the absence of urea. To confirm widespread protein carbamylation in rat IM, we carried out protein mass spectrometry. Proteins, processed in the absence of urea, were separated via SDS‐PAGE. Successive gel blocks were subjected to in‐gel trypsin digestion. Using LC‐MS/MS (Orbitrap) and a high‐stringency spectral search (SEQUEST), we identified 686 carbamylation sites in 579 proteins. Many of the identified sites were present on lysine side chains. The results show that extensive protein carbamylation occurs in rat renal IM, which could have an impact on cell signaling by blocking post‐translational modifications of lysines, e.g. acetylation and ubiquitylation.