Mass Spectrometry-Based\nDirect Sequencing of tRNAs\nDe Novo and Quantitative Mapping of Multiple RNA Modifications

Despite the extensive use of next-generation sequencing\n(NGS) of\nRNA, simultaneous direct sequencing and quantitative mapping of multiple\nRNA nucleotide modifications remains challenging. Mass spectrometry\n(MS)-based sequencing can directly sequence all RNA modifications\nwithout being limited to specific ones, but it requires a perfect\nMS ladder that few tRNAs can provide. Here, we describe an MS ladder\ncomplementation sequencing approach (MLC-Seq) that circumvents the\nperfect ladder requirement, allowing de novo MS sequencing of full-length\nheterogeneous cellular tRNAs with multiple nucleotide modifications\nat single-nucleotide precision. Unlike NGS-based methods, which lose\nRNA modification information, MLC-Seq preserves RNA sequence diversity\nand modification information, revealing new detailed stoichiometric\ntRNA modification profiles and their changes upon treatment with the\ndealkylating enzyme AlkB. It can also be combined with reference sequences\nto provide quantitative analysis of diverse tRNAs and modifications\nin total tRNA samples. MLC-Seq enables systematic, quantitative, and\nsite-specific mapping of RNA modifications, revealing the truly complete\ninformational content of tRNA.