Mass spectrometry-based detection of transfer RNAs by their signature endonuclease digestion products

Abstract

The separation of biologically active, pure, and specific tRNAs is difficult due to the overall similarity in secondary and tertiary structures of different tRNAs. Because prior methods do not facilitate high-resolution separations of the extremely complex mixture represented by a cellular tRNA population, global studies of tRNA identity and/or abundance are difficult. We have discovered that the enzymatic digestion of an individual tRNA by a ribonuclease (e.g., RNase T1) will generate digestion products unique to that particular tRNA, and we show that a comparison of an organism's complete complement of tRNA RNase digestion products yields a set of unique or ''signature'' digestion product(s) that ultimately enable the detection of individual tRNAs from a total tRNA pool. Detection is facilitated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and proof-of-principle is demonstrated on the whole tRNA pool from Escherichia coli. This method will enable the individual identification of tRNA isoacceptors without requiring specific affinity purification or extensive chromatographic and/or electrophoretic purification. Further, experimental identifications of tRNAs or other RNAs will now be possible using this signature digestion product approach in a manner similar to peptide mass fingerprinting used in proteomics, allowing RNomic studies of RNA at the post-transcriptional level.

Improving CMC-derivatization of pseudouridine in RNA for mass spectrometric detection

“…The contaminating tRNAs were found to be tRNA Trp (signature ions: m/z 1638, 1888, 2500, 2553 and 3944), tRNA Ser (signature ions: m/z 1349, 2289 and 2402) and tRNA His (signature ion: m/z 2425). It is apparent that this commercial E. coli tRNA TyrII sample also contains tRNA Trp , tRNA Ser and tRNA His , at a minimum, which is not surprising given the difficulty of purifying individual tRNAs [24]. These interfering tRNAs do not affect the ability to examine and optimize CMC derivatization conditions.…”

“…Theoretical RNase digestion products of E. coli tRNA TyrII were calculated using the Mongo Oligo Mass Calculator (http//www-medstat.med.utah.edu/massspec/mongo.htm). Signature digestion product lists were obtained from the RNAccess database (http://bearcatms.uc.edu/rnaccess/) [24]. MALDI peak lists were exported to Microsoft Excel for manipulation and analysis.…”

“…Table 1 RNase T1 digestion products of E. coli tRNA TyrII arising from data in Figure 2. Sequences listed are those that map to the known sequence of tRNA TyrII or tRNA signature products [24] …”

“…Several could be assigned as contaminating E. coli tRNAs through identification of their signature digestion products [24]. The contaminating tRNAs were found to be tRNA Trp (signature ions: m/z 1638, 1888, 2500, 2553 and 3944), tRNA Ser (signature ions: m/z 1349, 2289 and 2402) and tRNA His (signature ion: m/z 2425).…”

A eole for the MS analysis of nucleic acids in the post-genomics age

“…The potential of MS-technologies for large scale analysis of cellular RNAs has been demonstrated by the implementation of fingerprinting strategies based on endonuclease digestion of total tRNA extracts, followed by MALDI-MS detection of signature products ( Figure 4) [81,82]. Analogous to approaches employed in quantitative proteomics, 18 O end-labeling by nuclease digestion can be employed to evaluate RNA production and observe possible variations in the amount of posttranscriptionally modified nucleotides [83].…”