Poster abstracts
Poster number 117 submitted by Bibek Hamal
Optimization of RNase MC1 and Cusativin Digestion Protocols for tRNA Modification Mapping using LC-MS/MS
Bibek Hamal (Chemistry Department, University of Cincinnati), Hina Zain (Chemistry Department, University of Cincinnati), Christian Reidy Senior Manager, Genetic Medicine (Senior Manager, Genetic Medicine, Waters Corporation), Tatiana Johnston (Senior Scientist, Waters Corporation), Balasubrahmanyam Addepalli (Director, Waters Corporation)
Abstract:
tRNA modification mapping by mass spectrometry localizes modified nucleosides along intact sequences by generating overlapping oligonucleotides that are sequenced by liquid chromatography tandem mass spectrometry (LC-MS/MS). Modification mapping has been limited by the paucity of commercial RNases that generate MS-friendly oligonucleotides. To enhance modification mapping, we explored two new commercial offerings: MC1 and Cusativin. MC1 performs 5′ uridine cleavage at NpU sites (N = A, C, U) to create 2′,3′-cyclic-phosphate termini. MC1 also shows strong cleavage activity toward ([D]p[D]) and [m5C]pU but exhibits reduced activity when processing GpU. Cusativin cleaves cytidine bases from the 3′ end at CpN sites (N = U, A, G) to create 2′,3′-cyclic-phosphate termini. Cusativin showed high efficiency at Cp[m2,2G] and [m5C]pU but no activity at CpC.
We tested yeast tRNA Phenylalanine (78 nt; 9 modifications) at three different concentrations of 5, 10 and 15 U per µg to confirm the established cleavage rules. The highest sequence coverage along with less abundant modified digestion products emerged when using 15 U per µg of each enzyme. Using E. coli total tRNA (43 sequences with 27 modification types) showed MC1 reaching approx. 90% coverage and Cusativin reaching approx.90% coverage at this 15U per µg level. Using more complex S.cerevascae total tRNA ( 49 sequences with 23 modifications) showed MC1 reaching approx. 80% sequence coverage and Cusativin reaching approx. 90 % sequence coverage. These commercial enzymes should significantly enhance the use of LC-MS/MS for modification analyses.
Keywords: tRNA, Oligonucleotide