pH-Controlled chemoselective rapid azo-coupling reaction (CRACR) enables global profiling of serotonylation proteome in cancer cells

Serotonylation has been identified as a novel protein post-translational modification (PTM) for decades, where an isopeptide bond is formed between the glutamine residue and serotonin through transamination. Transglutaminase 2 (also known as TGM2 or TGase2) was proven to act as the main writer enzyme for this PTM and a number of key regulatory proteins (including small GTPases, fibronectin, fibrinogen, serotonin transporter, and histone H3) have been characterized as the substrates of serotonylation. However, due to the lack of pan-specific antibody for serotonylated glutamine, the precise enrichment and proteomic profiling of serotonylation still remain challenging. In our previous research, we developed an aryldiazonium probe to label protein serotonylation in a bioorthogonal manner. This chemical biology tool can be utilized alternatively for the antibody-free enrichment of serotonylated proteins, which depends on a pH-controlled chemoselective rapid azo-coupling reaction (CRACR). Here, we report the application of a photoactive aryldiazonium-biotin probe for the global profiling of serotonylation proteome in cancer cells. Thus, over 500 serotonylated proteins were identified from HCT 116 cells. Importantly, a number of modification sites of these serotonylated proteins were determine, attributed to the successful application of our chemical proteomic approach. Overall, these findings provided new insights into the significant association between cellular protein serotonylation and cancer development, further suggesting that to target TGM2-mediated monoaminylation may serve as a promising strategy for cancer therapeutics.