Limbach Group Biological Mass Spectrometry

Our current and future efforts are now directed toward establishing (1) More powerful mass spectrometry sequencing methods for RNAs and (2) Applying these sequencing tools to more complex collections of tRNAs, such as those found in human cancers. Our past and present research in de novo sequencing of tRNAs suggested that the inherent properties of tRNAs could be used to our advantage for tRNA sequencing. Prime amongst those properties is the relatively strong sequence and structural conservation of tRNAs (due to their fundamental role in protein translation). Thus, while those similarities generate significant challenges when one seeks to purify each constituent tRNA from all others in an organism, in our most recent research activities we seek to use those properties to our advantage. 

Comparative sequencing can be used for the sequencing of unknown tRNAs from host organisms whose genomes are known, for sequencing entirely unknown tRNAs if a suitable reference set of tRNAs exist, and is an ideal method to examine the specific effects of gene knockouts or knockdowns on tRNA sequence and modification. To take advantage of this new technology, we are now examining tRNA changes that occur in various eukaryotic cells - even when we do not know the entire modification status of every constituent tRNA, comparative sequencing allows us to easily identify which tRNAs are differentially expressed or modified (or both) between two sample types.