Polyacrylamide Gel Electrophoresis Coupled with Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for tRNA Mutant Analysis

摘要

In analogy to two-dimensional analysis, the mobility shift in native polyacrylamide gel electrophoresis (PAGE) due to a nucleotide substitution of a single-stranded transfer ribonucleic acid (tRNA) fragment serves as the first dimension for tRNA mutation analysis. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), as the second dimension, allows precise determination of the mass of the tRNA fragments resolved by native PAGE. Off-line combination of native PAGE with MALDI-MS is demonstrated for high-resolution analysis of tRNA↑(val) and its mutants, including a three-nucleotide deletion and 12 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 mere-bane, 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 pmol 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-trihydroxyacetophenone in acetone.