Delicate Analysis of Post-Translational Modifications on Dishevelled 3

The Wnt/β-catenin signaling pathway plays a critical
role
in multiple developmental events during embryogenesis and adult tissue
homeostasis. Dishevelled (Dvl) is an important component of Wnt/β-catenin
signaling pathway, although the modification, especially phosphorylation,
seems closely related to the activity and stability of Dvl, the overall
modification status of Dvl is still poorly understood. In this study,
we focused on low abundant Dvl3, one of the three Dvl isoforms. Using
affinity purification of different sources of Dvl3, followed by digestion
with both trypsin and chymotrypsin, we systematically analyzed the
overall modification status of Dvl3 with liquid chromatography coupled
LTQ-Orbitrap. Altogether, we confidently identified Dvl3 with more
than 50% sequence coverage, including 6 phosphorylation, 4 methylation,
and 1 dimethylation sites. Most of the identified modification sites
were novel and located in the linker region between different motifs.
Subsequently, the validity of modified peptides was confirmed by synthetic
modified peptides. Finally, SRM analysis was performed to verify and
quantify the successive change of one dimethylation site on Dvl3 in
vivo during Wnt signaling. Taken together, our study elucidated the
existence of a novel post-translational modification on Dvl3 and provided
a delicate experimental procedure for analysis of modification on
low abundant proteins.