Previous results for characterization of modified tRNAs by CAD revealed high-abundance w ions that appeared to be specific to a methylated guanine nucleobase present in the sequence of a yeast phenylalanine tRNA. Orthogonal sequencing approaches determined that this nucleobase was a 7-methylguanine, which contains a fixed positive charge, potentially enhancing this type of fragmentation. Previous MS-based studies also found facile base-loss of 7-methylguanine nucleobases upon collisional activation. Positional methylation isomers of adenine and cytidine, including 1-methyladenine and 3-methylcytidine, can likewise exhibit fixed positive charges, and thus might also be anticipated to enhance fragmentation. Of the tRNAs present in the T-Psi-C database, 26 are reported to contain at least one of these modifications (e.g., 7-methylguanine, 1-methyladenine, 3-methylcytidine). One of the most well-characterized of these tRNAs is Val IAC tRNA (tdbR00000464) noted as one of the six tRNAs in