Figure 4.

Tandem MS in positive ion mode of compound #6 comparing with the fragmentation pattern of m¹Ψ. Key fragments are indicated in boxes, and are seen to increase in mass by 16 Da showing O- to S- substitution. The fragmentation pattern is consistent with compound #6 being m¹s⁴Ψ. The positions of the methyl group at N1 and the sulphur on C4 of the pseudouridine were conclusively verified by fragmentation in the negative ion mode and comparison with additional standards (Figure 5).