Fig. 2: Identification of the ribosome-arresting peptidyl-tRNA by LC-MS/MS.
From: A mini-hairpin shaped nascent peptide blocks translation termination by a distinct mechanism
a Schematic illustration of the experimental procedure for the identification of peptidyl-tRNA-derived peptides. Each candidate sORF was overexpressed in E. coli cells, and the total RNA fraction in the cell lysate was concentrated with a silica column. The RNA fraction was hydrolyzed by alkaline treatment, and the obtained peptides were identified by protease digestion and subsequent LC-MS/MS. b–e MS/MS spectra of peptides derived from peptidyl-tRNAs. Amino acid sequences above the graphs represent the positions of the detected peptides (blue areas indicate the detected peptide). P and A below the amino acid sequence represent the plausible positions of the P- and A-sites in the ribosome during translation arrest, respectively. In the graphs, the peaks of the b- and y-fragment ions are shown in red and blue, respectively.
