Table 1. Methods used in the discovery and characterization of RNA modifications (2009-2014).

Identity	Symbol	Structure	RNA Species	Discovery	Validation	Quantification
5-Cyanomethyluridine	cnm5U		tRNA	Targeted analysis of mutant phenotype	Std, pMS3	MRM
N6-Hydroxymethyladenosine	hm6A		mRNA	Targeted analysis of modified synthetic ssRNA	Std, MS2	MRM
N6-Formyladenosine	f6A		mRNA	Targeted analysis of modified synthetic ssRNA	Std, MS2	MRM
Cyclic N6 Threonylcarbamoyl-adenosine	t6A		tRNA	Targeted analysis of natural product	Std, MSn, 18O-labeling, NMR	UV absorbance
2-Geranylthiouridines*	ge5s2U		tRNA	LC-MS/MS screen of nucleotides	Std, MS2, 13C-labeling	External calibration
N6, N6-dimethyladenosine	m6,6A		tRNA	LC-MS/MS screen of nucleotides	Std, pMS3	MRM
2,8-Dimethyladenosine	m2,8A		rRNA	Targeted analysis of mutant phenotype	Std, MSn	Not performed
N-ribosylnicotinamide	r-NA		5′cap of sRNAs (>200 nt)	LC-MS/MS screen of nucleotides	Std, MS2, 13C-labeling	External calibration, 13C/15N-labeled isotope dilution
Co-enzyme A**	coA		5′cap of sRNAs (>200 nt)	LC-MS/MS screen of nucleotides	Std, MS2, 18O-labeling	External calibration

^*R₁ can be hydrogen, methylaminomethyl- or carboxymethylaminomethyl- groups (Dumelin et al., 2012);

^**R₂ can be hydrogen, succinyl-, acetyl- or methylmalonyl- groups (Kowtoniuk, Shen, Heemstra, Agarwal, & Liu, 2009); Std, synthetic standards; pMS3, pseudo-MS3 by controlled in-source fragmentation; MSn, tandem in-time fragmentation on ion-trap mass spectrometers.