Proteome-wide quantitative profiling of mis-incorporation of selenocysteine into proteins by LC-MS based shotgun proteomics

Abstract

It is well-known that selenocysteine (Sec) could be specifically incorporated into growing polypeptides, as dened by the stop codon UGA together with SECIS sequence elements. As a structural analog, however, Sec could compete for the tRNA^Cys against cysteine inserting into protein directly in place of cysteine. This UGA-independent mis-incorporation of Sec substituting cysteine, has yet to be studied on a proteome level, and the features of Sec mis-incorporation sites remains unclear. This research aims to profile the E. coli proteome for Sec mis-incorporation by using label-free quantitative proteomics methods. Taking advantage of LC-MS based shotgun proteomics technology, a total of 44 selenopeptides have been identified from cells exposed to a high-selenium medium. Analysis of the corresponding gene sequences for the identified selenopeptides showed that all the replaced cysteines were encoded by UGU or UGC instead of UGA, and the SECIS element was not found, indicating that the cysteines were replaced by Sec in an apparent UGA-independent manner. Interestingly, the mis-incorporation sites did not distribute randomly as expected. Across the proteome, some cysteine residues were consistently replaced whereas other cysteines were not replaced at all. Secondary structure analysis led to a possible explanation that mis-incorporation at critical sites caused misfolding and degradation of proteins making it undetectable in LC-MS. Quantitative comparative analysis between normal group and the high-selenium group revealed that the expression levels of 320 proteins have changed significantly, 251 of which have been down-regulated. Gene ontology and pathway analysis indicated that most of these proteins are involved in the biosynthesis of amino acids, tRNA aminoacylation and oxidation-reduction reactions. Proteins related to UGA-dependent incorporation of Sec were extensively down-regulated, which implied that a high concentration of Sec possibly has an inhibitory effect on the UGA-dependent pathway.

Date of Award	2016
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology