Polyacrylamide gel electrophoresis coupled with matrix-assisted laser desorption/ionization mass spectrometry for tRNA

Abstract

In analogy to two-dimensional analysis, the mobility shift in native polyacrylamide gel electrophoresis (PAGE) due to a nucleotide substitution of a single-stranded RNA fragment serves as the first dimension for RNA mutation analysis. Matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), as the second dimension, allows precise mass detennination of RNA fragments resolved by native PAGE. Off-line combination of native PAGE with MALDI-MS is demonstrated for high resolution analysis of tRNAᵛᵃˡ and its mutants, including a three nucleotide deletion and twelve single base substitutions. Three approaches, including direct extraction of tRNAs from gel into buffer solution, dissolution of membrane in the matrix solution, and direct desorption of tRNAs from the membrane, are studied for coupling native PAGE with MALDI-MS. The membrane dissolution method is simple and the resulting mixture is amenable to MALDI-MS analysis. In the membrane dissolution method, as little as 1 μg or 40 pmole of tRNA sample is loaded on a native gel, separated, capillary eluted onto a nitrocellulose membrane, and recovered using the matrix solution of 2,4,6-THAP in acetone.