Using engineered initiator tRNA for precise control of protein translation within cells has great promise within future orthogonal translation systems to decouple housekeeping protein metabolism from that of engineered genetic systems. Previously, E. coli strain C321.ΔA.exp lacking all UAG stop codons was created, freeing this ‘amber’ stop codon for other purposes. An engineered ‘amber initiator’ that activates translation at UAG codons is available, but little is known about amino acid it assigns to the amber start codon. Here, we combine for the first time the amber initiator tRNA in C321.ΔA.exp and measure its cellular effects. Using, targeted proteomics, we found that the amber initiator tRNA initiates proteins exclusively with methionine.

Parallel reaction monitoring (PRM) mass spectrometry method was used to selectively monitor peptides of nanoluciferase reporter protein that initiates either from a canonical (AUG) or an amber (UAG) start codon when amber initiator tRNA is expressed in the cell. The aim of the experiment was to determine the N-terminal amino acid attached to the nanoluciferase reporter protein. Samples were analysed on a high resolution Q-Exactive mass spectrometer (ThermoFischer Scientific) coupled to an EASY-nLC1000 liquid chromatography system. Peptides were eluted over a 120-minute linear gradient with increasing concentration of elution buffer (99.9% (v/v) acetonitrile, 0.1% formic acid). The analysis consisted of one survey (full) scan at 70,000 resolution (400 m/z) for targeted precursors (Table S10) (AGC target 2e5, maximum injection time 100ms, 2 m/z isolation width). Measured precursor ions were selected for HCD fragmentation with normalized collision energy of 30 followed by full ms/ms of product ions at 17,500 resolution (m/z 200, AGC target of 2e5 and 60ms maximum injection time). Precursor and fragment ion spectra were analysed using Skyline version 4.2.

ATG1 - Nickel NTA-column purified nanoluciferase protein with an ATG start codon (1st biological replicate). ATG2 - Nickel NTA-column purified nanoluciferase protein with an ATG start codon (2nd biological replicate). ATG3 - Nickel NTA-column purified nanoluciferase protein with an ATG start codon (3rd biological replicate). TAG1 - Nickel NTA-column purified nanoluciferase protein with a TAG start codon (1st biological replicate). TAG2 - Nickel NTA-column purified nanoluciferase protein with a TAG start codon (2nd biological replicate). TAG3 - Nickel NTA-column purified nanoluciferase protein with a TAG start codon (3rd biological replicate).