Recent studies revealed that the 3'-terminal nucleotides in plant microRNAs were methylated on the ribose at the 2' or 3' hydroxyl groups. Here we examined the fragmentation of the electrospray-produced [M + H]+ and [M - H]- ions of 2'- and 3'-O-methylated ribonucleosides. It turned out that the predominant fragmentation pathway for the [M + H]+ ions of ribose-methylated nucleosides was the neutral loss of the methylated ribose, which made it impossible to distinguish 2'-O-methylation from 3'-O-methylation by positive-ion MS/MS. However, characteristic fragment ions, resulting from the cleavage through the ribose rings, were produced for the [M - H]- ions of each pair of ribose-methylated nucleosides. In this respect, the neutral loss of a 90-Da fragment (C3H6O3) was observed for 2'-O-methylated cytidine, guanosine and adenosine, but not for their 3'-O-methylated counterparts. On the other hand, the neutral loss of a 60-Da fragment (C2H4O2) was found for 3'-O-methyluridine, but not for 2'-O-methyluridine.
