Mass Spectrometry-Based Validation of Chimeric RNAs.

Chimeric RNAs, formed by the fusion of exons from two or more distinct genes, represent a significant class of noncanonical transcripts with increasing implications in cancer biology, development, and other biological processes. Their inherent novelty and the potential for sequence similarity with parental transcripts pose significant challenges for accurate detection and validation. While next-generation sequencing (NGS) has become the primary tool for chimeric RNA discovery, orthogonal validation methods are crucial to confirm their existence, delineate their precise structure, and quantify their abundance. Mass spectrometry (MS)-based approaches offer a powerful and complementary strategy for the robust validation of chimeric RNAs. This chapter will delve into the principles and applications of MS-based techniques for the definitive characterization of these fusion transcripts, highlighting their strengths in providing direct evidence of the chimeric junction at the peptide level, confirming the reading frame, and offering quantitative insights. We will explore various MS workflows, including targeted and untargeted peptidomics, and discuss the critical considerations for sample preparation, data acquisition, and bioinformatic analysis to ensure reliable and high-confidence validation of chimeric RNAs.