Mass Spectrometry-Based Methods for Characterization of Hypomodifications in Transfer RNA

Quantitative characterization of the identity and location of nucleoside modifications (added posttranscriptionally) in RNA molecules is the first step for associating them to biochemical functions and disease. This chapter examines the ways to reduce the RNA complexity for accurate identification and quantification of modifications in transfer RNA (tRNA). These analytical methods are organized under three subsections. (1) RNA purification: We will contrast the classical and the affinity-based purification methods for tRNA enrichment. (2) Modification detection and abundance: Liquid chromatography coupled with mass spectrometry (LC-MS) methods involving high-energy collision dissociation and chromatographic retention time-based assignment will be compared. Ways to compute the relative abundance and application of multiple mass filters to document absolute amounts with external vs internal calibration for computing changes will be discussed. (3) Site-specific quantification: Relative quantification of modifications in the sequence context through isotope-labeling and signature oligonucleotide digestion products during RNA modification mapping will be examined. Thus, we will discuss a range of methods suitable for characterization of hypomodifications in tRNA (or any other RNA) in this chapter. [GRAPHICS] .