Quantitative Proteomics Reveals UGA-Independent Misincorporation of Selenocysteine throughout the Escherichia coli Proteome

Selenocysteine
is cotranslationally inserted into polypeptide chains
by recoding the stop codon UGA. However, selenocysteine has also been
found to be misincorporated into a small number of proteins displacing
cysteines in previous studies, but such misincorporation has not yet
been examined at the proteome level thoroughly. We performed label-free
quantitative proteomics analysis on Escherichia coli grown in a high-selenium medium to obtain a fuller picture of selenocysteine
misincorporation in its proteome. We found 139 misincorporation sites,
including 54 recurred in all biological replicates, suggesting that
some cysteine sites are more prone to be misincorporated than others.
However, sequence and evolutionary conservation analysis showed no
clear pattern among these misincorporation sites. We hypothesize that
misincorporations occur randomly throughout the proteome, but the
degradation rate of such misincorporated proteins varies depending
on the impact of the misincorporation on protein function and stability,
leading to the differential detectability of misincorporated sites
by proteomics. Our hypothesis is further supported by two observations:
(1) cells cultured with severely limited sulfur still retained a substantial
proportion of normal cysteine counterparts of all of the found misincorporated
proteins and (2) proteins involved in protein folding and proteolysis
were highly upregulated in high-selenium culture.