Synaptic Protein Ubiquitination in Rat Brain Revealed by Antibody-based Ubiquitome Analysis

Protein ubiquitination is an essential post-translational
modification
regulating neurodevelopment, synaptic plasticity, learning, and memory,
and its dysregulation contributes to the pathogenesis of neurological
diseases. Here we report a systematic analysis of ubiquitinated proteome
(ubiquitome) in rat brain using a newly developed monoclonal antibody
that recognizes the diglycine tag on lysine residues in trypsinized
peptides (K-GG peptides). Initial antibody specificity analysis showed
that the antibody can distinguish K-GG peptides from linear GG peptides
or pseudo K-GG peptides derived from iodoacetamide. To evaluate the
false discovery rate of K-GG peptide matches during database search,
we introduced a null experiment using bacterial lysate that contains
no such peptides. The brain ubiquitome was then analyzed by this antibody
enrichment with or without strong cation exchange (SCX) prefractionation.
During SCX chromatography, although the vast majority of K-GG peptides
were detected in the fractions containing at least three positive
charged peptides, specific K-GG peptides with two positive charges
(e.g., protein N-terminal acetylated and C-terminal non-K/R peptides)
were also identified in early fractions. The reliability of C-terminal
K-GG peptides was also extensively investigated. Finally, we collected
a data set of 1786 K-GG sites on 2064 peptides in 921 proteins and
estimated their abundance by spectral counting. The study reveals
a wide range of ubiquitination events on key components in presynaptic
region (e.g., Bassoon, NSF, SNAP25, synapsin, synaptotagmin, and syntaxin)
and postsynaptic density (e.g., PSD-95, GKAP, CaMKII, as well as receptors
for NMDA, AMPA, GABA, serotonin, and acetylcholine). We also determined
ubiquitination sites on amyloid precursor protein and alpha synuclein
that are thought to be causative agents in Alzhermer’s and
Parkinson’s disorders, respectively. As K-GG peptides can also
be produced from Nedd8 or ISG15 modified proteins, we quantified these
proteins in the brain and found that their levels are less than 2%
of ubiquitin. Together, this study demonstrates that a large number
of neuronal proteins are modified by ubiquitination and provides a
feasible method for profiling the ubiquitome in the brain.