Revealing the amino acid composition of proteins within an expanded genetic code. Nucleic Acids Res

The genetic code can be manipulated to reassign codons for the incorporation of non-standard amino acids (NSAA). Deletion of release factor 1 in Es-cherichia coli enhances translation of UAG (Stop) codons, yet may also extended protein synthesis at natural UAG terminated messenger RNAs. The fi-delity of protein synthesis at reassigned UAG codons and the purity of the NSAA containing proteins produced require careful examination. Proteomics would be an ideal tool for these tasks, but con-ventional proteomic analyses cannot readily iden-tify the extended proteins and accurately discover multiple amino acid (AA) insertions at a single UAG. To address these challenges, we created a new pro-teomic workflow that enabled the detection of UAG readthrough in native proteins in E. coli strains in which UAG was reassigned to encode phosphoser-ine. The method also enabled quantitation of NSAA and natural AA incorporation at UAG in a recom-binant reporter protein. As a proof-of-principle, we measured the fidelity and purity of the phosphoser-ine orthogonal translation system (OTS) and used this information to improve its performance. Our re-sults show a surprising diversity of natural AAs at reassigned stop codons. Our method can be used to improve OTSs and to quantify amino acid purity at reassigned codons in organisms with expanded genetic codes