Characterization of Protein Serotonylation via Bioorthogonal Labeling and Enrichment

Abstract

Protein serotonylation is a transglutaminase-mediated phenomenon whose biological mechanism of protein serotonylation is not yet fully understood, as the complete profiling of serotonylation targets in a proteome remains a critical challenge to date. Utilizing an alkyne-functionalized serotonin derivative bioorthogonally coupled to a cleavable linker, we developed a method to selectively enrich serotonylated proteins in a complex sample. With online nanoflow liquid chromatography and LTQ-Orbitrap Velos hybrid mass spectrometer detection, we identified 46 proteins with 50 serotonylation sites at their glutamine residues. Mass spectrometric analysis also generated direct residue-level evidence of various biological processes such as transglutaminase-chaperon interactions as well as actin assembly. An enrichment workflow utilizing click chemistry and on-bead digestion allowed us to achieve site-specific identification of protein serotonylation by mass spectrometry, and results obtained hereby also provided a great foundation in the elucidation of the true roles of protein serotonylation in biological systems.

Histone serotonylation is a permissive modification that enhances TFIID binding to H3K4me3

“…Biotin cadaverine was purchased from Invitrogen. 5-PT was prepared in house as a hydrochloride salt according to reported synthetic procedures 15 .…”

“…Given that TGM2 may interact with, or even cross-link, histone proteins in vitro 12–14 , we began by investigating whether histones might serve as endogenous substrates for serotonylation. We employed a bioorthogonal metabolic labeling approach 15 based on propargylated serotonin (5-PT) that can be delivered to living cells (Extended Data Fig. 1a-b), followed by protein labeling and immunoprecipitation (IP) of serotonylated substrates (Fig.…”

Serotonylation: Serotonin Signaling and Epigenetics

“…However, despite that mass spectrometry was performed the serotonylated Gln residues in these proteins were not reported which would have supported the specificity of the labeling method using biotinylated serotonin. Notwithstandingly, at least some of the discovered proteins, such as actin, were confirmed in other studies using propargylated serotonin or MDC and mass spectrometry in different cell types (Lin et al, 2014; Hummerich et al, 2015).…”

“…Moreover the molecule gets very large and can not enter cells anymore. Therefore biorthogonal labeling was developed which used a small alkyne-functionalized serotonin, which can be taken up in cells for the transglutaminase reaction (Lin et al, 2013, 2014). Afterwards a biotin-residue is specifically linked by click-chemistry to this incorporated propargylated serotonin.…”

“…Afterwards a biotin-residue is specifically linked by click-chemistry to this incorporated propargylated serotonin. The biotin-label then allows purification and detection of the serotonylated protein on Western blots and by mass spectrometry (Lin et al, 2013, 2014; Farrelly et al, 2019).…”