pH-Controlled\nChemoselective Rapid Azo-Coupling Reaction\n(CRACR) Enables Global Profiling of Serotonylation Proteome in Cancer\nCells

Serotonylation has been identified as a novel protein\nposttranslational\nmodification for decades, where an isopeptide bond is formed between\nthe glutamine residue and serotonin through transamination. Transglutaminase\n2 (also known as TGM2 or TGase2) was proven to act as the main “writer”\nenzyme for this PTM, and a number of key regulatory proteins (including\nsmall GTPases, fibronectin, fibrinogen, serotonin transporter, and\nhistone H3) have been characterized as the substrates of serotonylation.\nHowever, due to the lack of pan-specific antibodies for serotonylated\nglutamine, the precise enrichment and proteomic profiling of serotonylation\nstill remain challenging. In our previous research, we developed an\naryldiazonium probe to specifically label protein serotonylation in\na bioorthogonal manner, which depended on a pH-controlled chemoselective\nrapid azo-coupling reaction. Here, we report the application of a\nphotoactive aryldiazonium-biotin probe for the global profiling of\nserotonylation proteome in cancer cells. Thus, over 1,000 serotonylated\nproteins were identified from HCT 116 cells, many of which are highly\nrelated to carcinogenesis. Moreover, a number of modification sites\nof these serotonylated proteins were determined, attributed to the\nsuccessful application of our chemical proteomic approach. Overall,\nthese findings provided new insights into the significant association\nbetween cellular protein serotonylation and cancer development, further\nsuggesting that to target TGM2-mediated monoaminylation may serve\nas a promising strategy for cancer therapeutics.