5305 vacufuge plus concentrator

To identify the accumulation of D-aa-tRNA adducts, overnight grown primary culture of DTD1 knockout E. coli was used to inoculate 1% secondary culture in minimal media with or without 2.5 mM D-tyrosine. Secondary culture grown to OD₆₅₀ (optical density at 650 nm) 0.8 was subjected to respective aldehyde treatment (0.01% final concentration) with 0.5 mM NaCNBH₃ at 37°C for 30 min. Cultures were pelleted and total RNA was isolated through acidic phenol chloroform method. Total RNA was digested with three volumes of aqueous ammonia (25% of vol/vol NH₄OH) at 70°C for 18 hr (Mazeed et al., 2021 (link)). Hydrolysed samples were dried using Eppendorf 5305 Vacufuge plus Concentrator. Dried samples were resuspended in 10% methanol and 1% acetic acid in water and analysed via ESI-based mass spectrometry using a Q-Exactive mass spectrometer (Thermo Scientific) as mentioned above.

To identify the modification by various aldehydes on D-aa-tRNAs, modified and unmodified D-Phe-tRNA^Phe were digested with aqueous ammonia (25% of vol/vol NH₄OH) at 70°C for 18 hr (Mazeed et al., 2021 (link)). Hydrolysed samples were dried using Eppendorf 5305 Vacufuge plus Concentrator. Dried samples were resuspended in 10% methanol and 1% acetic acid in water and analysed via ESI-based mass spectrometry using a Q-Exactive mass spectrometer (Thermo Scientific) by infusing through heated electrospray ionisation source operating at a positive voltage of 3.5 kV. Targeted selected ion monitoring (t-SIM) was used to acquire the mass spectra (at a resolving power of 70,000@200 m/z) with an isolation window of 2 m/z, i.e., theoretical m/z and MH+ ion species. The high energy collision-induced MS-MS spectra with a normalised collision energy of 25 of the selected precursor ion species specified in the inclusion list (having the observed m/z value from the earlier t-SIM analysis) were acquired using the method of t-SIM-ddMS2 (at an isolation window of 1 m/z at a ddMS2 resolving power of 35,000@200 m/z).

A single-step method was used for probing relative modification propensity of the aldehyde with aa-tRNA where 0.2 µM of Ala-tRNA^Ala was incubated with different concentrations of aldehydes (2 mM and 10 mM) along with 20 mM NaCNBH₃ (in 100 mM potassium acetate [pH 5.4]) as a reducing agent at 37°C for 30 min. The reaction mixture was digested with S1 nuclease and analysed on TLC. Except for decanal, all the aldehydes modified Ala-tRNA^Ala. The method for processing and quantification of modification on aa-tRNA utilised is discussed earlier (Mazeed et al., 2021 (link)). However, a two-step method was used for generating substrates for biochemical assays as discussed earlier (Mazeed et al., 2021 (link)). It was used to generate maximum homogenous modification on the aa-tRNAs for deacylation assays. Briefly, 2 µM aa-tRNAs were incubated with 20 mM of formaldehyde, and methylglyoxal or 1 M of propionaldehyde, butyraldehyde, valeraldehyde, and isolvaleraldehyde at 37°C for 30 min. Samples were dried to evaporate excess aldehydes using Eppendorf 5305 Vacufuge plus Concentrator. The dried mixture was then reduced with 20 mM NaCNBH₃ at 37°C for 30 min. All reactions were ethanol-precipitated at –30°C overnight or –80°C for 2 hr. Ethanol precipitated pellets were resuspended in 5 mM sodium acetate (pH 5.4) and used for biochemical assays.