Figure 2.

SILNAS-based PTM analysis. (A) Ψ76 in S. pombe 5.8S rRNA. (a) Extracted ion monitoring of RNase T1 fragments of 5.8S rRNA containing PTMs. Because PTM of U76 was incomplete (76% as estimated by SILNAS), RNase T1 produced two fragments, ⁷⁴AAΨU⁷⁸Gp and ⁷⁴AAUU⁷⁸Gp, from the sequence region 74–78 of 5.8S rRNA. In a subsequent LC analysis, the fragment ⁷⁴AAΨU⁷⁸Gp eluted earlier than the unmodified fragment ⁷⁴AAUU⁷⁸Gp and the corresponding heavy fragment ⁷⁴AAUU⁷⁸*Gp (*G, 13C10-guanosine) derived from the reference RNA. Oligonucleotide ion masses: [AAUUGp]²⁻ and [AAΨUGp]²⁻ (black line), m/z 815.60; [AAUU*Gp]²⁻ (red line), m/z 820.62. (b) MS spectrum for each RNA peak, (i) and (ii). From the signal heights of MS spectra of the light and heavy AAUUGp ion, the extent of PTM was estimated as 76%. (B) Ψ1039 in S. pombe 18S rRNA. (a) Extracted ion monitoring of RNase T1 fragments of 18S rRNA containing PTMs. Although PTM of U1039 was almost complete (99.7% as estimated by SILNAS), a trace amount of unmodified RNase T1 fragment, ¹⁰³⁰UCUUAACCAUAAACUAU¹⁰⁴⁷Gp, was detected with the modified fragment ¹⁰³⁰UCUUAACCAΨAAACUAU¹⁰⁴⁷Gp from the sequence region 1030–1047 of 18S rRNA. In a mass chromatogram, the modified fragment ¹⁰³⁰UCUUAACCAΨAAACUAU¹⁰⁴⁷Gp eluted later than the unmodified fragment ¹⁰³⁰UCUUAACCAUAAACUAU¹⁰⁴⁷Gp and the corresponding heavy fragment ¹⁰³⁰UCUUAACCAUAAACUAU¹⁰⁴⁷*Gp (*G, ¹³C₁₀-guanosine) derived from the reference RNA. Oligonucleotide ion masses: [UCUUAACCAUAAACUAUGp]⁴⁻ and [UCUUAACCAΨAAACUAUGp]⁴⁻ (black line), m/z 1429.67; [UCUUAACCAUAAACUAU*Gp]⁴⁻ (red line), m/z 1432.18. The mass windows used for extraction were 15 ppm. (b) MS spectrum for each RNA peak, (i) and (ii). From the signal heights of MS spectra of the light and heavy UCUUAACCAUAAACUAUGp ion, the extent of PTM was estimated as 99.7%.