Tendinous ruptures in rheumatoid arthritis.

Abstract

<h3>Abstract</h3> Life depends on proteins, which all exist in nascent states when the growing polypeptide chain is covalently attached to a tRNA within the ribosome. Although the nascent chains; <i>i</i>.<i>e</i>., polypeptidyl-tRNAs (pep-tRNAs), are considered as merely transient intermediates during protein synthesis, recent advances have revealed that they are directly involved in a variety of cell functions, such as gene expression control. An increasing appreciation for fine-tuning at translational levels demands a general method to handle the pep-tRNAs on a large scale. Here, we developed a method termed <i>pe</i>ptidyl-<i>t</i>RNA <i>e</i>nrichment using <i>o</i>rganic extraction and <i>s</i>ilica adsorption (PETEOS), and then identify their polypeptide moieties by mass spectrometry. As a proof-of-concept experiment using <i>Escherichia coli</i>, we identified ∼800 proteins derived from the pep-tRNAs, which were markedly biased towards the N-termini in the proteins, reflecting that PETEOS captured the intermediate pep-tRNA population during translation. Furthermore, we observed the changes in the pep-tRNA set in response to heat shock or antibiotic treatments. In summary, PETEOS will complement conventional methods for profiling nascent chains such as ribosome profiling. <h3>Significance Statement</h3> In the central dogma of biology, RNA and protein are usually regarded as two completely independent molecular species. However, they are combined into a single species called peptidyl-tRNA (pep-tRNA) during the translation process in the ribosome. Despite the importance of pep-tRNAs as precursors of all proteins in the cell, a general method to analyze pep-tRNAs on a large scale was lacking. Taking advantage of the properties of pep-tRNAs as RNA and protein, we developed a method to enrich the pep-tRNAs by organic solvent extraction and silica column separation. The method, termed PETEOS, not only provides a unique approach to examine the nascent state of proteins but also may be effective in capturing snapshots of translation status in the cell.