Differentiation of 2′-O- and 3′-O-methylated Ribonucleosides by tandem mass spectrometry
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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.
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