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.

FIGURE 1.

MALDI mass spectra obtained from the RNase T1 digestion of mixtures of E. coli tRNAs. (A) tRNAs Tyr I, Val III, and Glu II. (B) tRNAs Phe, Val III, and Glu II. (C) tRNAs Tyr I, Phe, and Glu II. (D) tRNAs Tyr I, Phe, and Val III. RNase T1 signature digestion products for each tRNA are labeled: (Δ) Tyr I; (□) Phe; (•) Val III; and (○) Glu II. (E) Heat map representation of this data, along with m/z and sequences of signature digestion products. In all analyses, reproducible detection of the signature digestion products of the expected tRNAs is demonstrated.

FIGURE 2.

MALDI mass spectra obtained from the RNase T1 digestion of E. coli tRNAs. (A) m/z 900–2700; (B) m/z 2700–6000. (C) MALDI mass spectra obtained from the RNase A digestion of E. coli tRNAs. Signature products are labeled with assignments listed in Tables 3 and 4 for RNases T1 and A, respectively.

FIGURE 3.

Signature digestion product coverage of tRNA families analyzed from an E. coli cell lysate using RNases T1 and A.