Data-Independent Acquisition for the Detection of Mononucleoside RNA Modifications by Mass Spectrometry

RNA is dynamically
modified in cells by a plethora of chemical
moieties to modulate molecular functions and processes. Over 140 modifications
have been identified across species and RNA types, with the highest
density and diversity of modifications found in tRNA (tRNA). The methods
used to identify and quantify these modifications have developed over
recent years and continue to advance, primarily in the fields of next-generation
sequencing (NGS) and mass spectrometry (MS). Most current NGS methods
are limited to antibody-recognized or chemically derivatized modifications
and have limitations in identifying multiple modifications simultaneously.
Mass spectrometry can overcome both of these issues, accurately identifying
a large number of modifications in a single run. Here, we present
advances in MS data acquisition for the purpose of RNA modification
identification and quantitation. Using this approach, we identified
multiple tRNA wobble position modifications in Arabidopsis
thaliana that are upregulated in salt-stressed growth conditions
and may stabilize translation of salt stress induced proteins. This
work presents improvements in methods for studying RNA modifications
and introduces a possible regulatory role of wobble position modifications
in A. thaliana translation.