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
posttranslational
modification 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 antibodies for serotonylated
glutamine, the precise enrichment and proteomic profiling of serotonylation
still remain challenging. In our previous research, we developed an
aryldiazonium probe to specifically label protein serotonylation in
a bioorthogonal manner, which depended on a pH-controlled chemoselective
rapid azo-coupling reaction. Here, we report the application of a
photoactive aryldiazonium-biotin probe for the global profiling of
serotonylation proteome in cancer cells. Thus, over 1,000 serotonylated
proteins were identified from HCT 116 cells, many of which are highly
related to carcinogenesis. Moreover, a number of modification sites
of these serotonylated proteins were determined, 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.