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

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 tRNAval 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 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 microg 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.