Figure 3.

Mass spectrometric analysis and quantification of full-length and reinitiated peptides in incorporation of two consecutive d-alanines. (A) mRNA and expressed peptide sequences: full-length peptide (FLPS2a) and reinitiated peptides (RiPS1a and RiPS2). (B) Identification of translation products by MALDI-TOF MS. Peptides were expressed with 0.03, 0.26 or 3.0 μM EF-G. ‘Calc.’ and ‘Obs.’ indicate calculated and observed m/z values, respectively. (C) Titration of EF-G concentration in translation of mRNAS2. RiPS1a and RiPS2 could not be separated on the tricine-SDS-PAGE gel due to the only single amino acid difference between them. (D) Titration of EF-G concentration in translation of mRNAS2. Expressed peptides were radioisotope-labelled by [¹⁴C]-Asp in the C-terminal FLAG sequences, separated by tricine-SDS-PAGE, and quantified by autoradiography. Yields of peptides were quantified by autoradiography. n = 3.