Targeted Quantitative Proteomics Revealed Profound Perturbations of the Epitranscriptome upon Genetic Depletion of TRUB1 Pseudouridine Synthase.

The study investigates how the genetic depletion of the TRUB1 enzyme (which installs Ψ55 on tRNAs) impacts the expression of other RNA-modifying proteins (readers, writers, and erasers) in human cells. Using targeted proteomics (LC-PRM) and SILAC, the researchers found that loss of TRUB1 leads to a significant reduction in the protein TARBP1 and a subsequent ~50% decrease in 2’-O-methylation of guanosine (Gm) levels in total tRNA. Cell Lysis: TRUB1 KO and parental HEK293T cells were cultured in SILAC media, harvested, and lysed using CelLytic M reagent with protease inhibitors. Protein Preparation: Protein concentrations were determined by Bradford assay. Heavy and light lysates (25 μg each) were combined and denatured with 8 M urea in a 30 kDa centrifugal filter. Digestion: Samples were reduced with dithiothreitol (DTT), alkylated with iodoacetamide, and digested overnight at 37°C with MS-grade trypsin at a 1:50 ratio. Clean-up: Tryptic peptides were desalted using C18 tips and redissolved in 0.1% formic acid for analysis. Mass Spectrometry: Tryptic peptides were analyzed using an LC-PRM method on a Fusion Lumos Orbitrap mass spectrometer equipped with FAIMS (Field Asymmetric-waveform Ion Mobility Spectrometry). Software: Data were imported into Skyline (Version 21.2) for analysis. Quantification required specific criteria: dotp > 0.7, rdotp > 0.7, and at least 4 fragment ions with co-eluting light and heavy species. Statistical Analysis: Chromatographic peak areas were processed using a customized R script (version 4.3.1) utilizing packages such as tidyverse, ggplot2, and scales.