February 15, 2024
Journal Article

Analysis of a macrophage carbamylated proteome reveals a function in post-translational modification crosstalk


Background. Lysine carbamylation is a biomarker of rheumatoid arthritis and kidney diseases. However, its cellular function is understudied due to the lack of tools for systematic analysis of this post-translational modification (PTM). Methods. We adapted a method to analyze carbamylated peptides by co-affinity purification with acetylated peptides based on the cross-reactivity of anti-acetyllysine antibodies. We integrated this method into a mass spectrometry-based multi-PTM pipeline to simultaneously analyze carbamylated and acetylated peptides in addition to phosphopeptides were enriched by sequential immobilized-metal affinity chromatography. Results. By testing the pipeline with RAW 264.7 macrophages treated with bacterial lipopolysaccharide, 7,299, 8,923 and 47,637 acetylated, carbamylated, and phosphorylated peptides were identified, respectively. Our analysis showed that carbamylation occurs on proteins from a variety of functions on sites with similar as well as distinct motifs compared to acetylation. To investigate possible PTM crosstalk, we integrated the carbamylation data with acetylation and phosphorylation data, leading to the identification 1,183 proteins that were modified by all 3 PTMs. Among these proteins, 54 had all 3 PTMs regulated by lipopolysaccharide and were enriched in immune signaling pathways, and in particular, the ubiquitin-proteasome pathway. We found that carbamylation of linear diubiquitin blocks the activity of the anti-inflammatory deubiquitinase OTULIN. Conclusions. Overall, our data show that anti-acetyllysine antibodies can be used for effective enrichment of carbamylated peptides. Moreover, carbamylation may play a role in PTM crosstalk with acetylation and phosphorylation, and that it is involved in regulating ubiquitination in vitro.

Published: February 15, 2024


You Y., C. Tsai, R. Patel, S. Sarkar, G. Clair, M. Zhou, and T. Liu, et al. 2023. Analysis of a macrophage carbamylated proteome reveals a function in post-translational modification crosstalk. Cell Communication and Signaling 21, no. 1:241. PNNL-SA-186292. doi:10.1186/s12964-023-01257-3

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