Acetic acid as mobile phase additive facilitated the identification and quantification of uridine modifications in negative ion mode by HPLC-MS/MS

RNA modifications are vital for all living organisms. A total of at least 63 types of uridine modification have been discovered, accounting for the largest proportion of all reported RNA modifications. Uridine modifications have also been shown to be dysregulated in various human diseases. However, it's well known that mass spectrometry analysis of uridine modifications is limited by their low ionization efficiencies. Here, we developed a simple and robust method for the simultaneous identification and quantification of 22 uridine modifications in biological samples. In fact, uridine and lots of its modifications are more easily ionized in negative ion mode, compared with positive ion mode commonly used. Besides, the fragmentation patterns of uridine modifications in negative ion mode could provide more structural information, compared with well-known glycosidic bond cleavage in positive ion mode. Furthermore, simply by adding 0.2 mM acetic acid into the mobile phase, the peak areas of uridine and all 22 uridine modifications in negative ion mode could be improved by 4.1-55.4 folds. With this novel method, we quantified 16, 14, 10, 12 and 16 uridine modifications in 293T total RNA, 293T small RNA, E. coli tRNA, S. cerevisiae tRNA and wheat germ tRNA, accordingly. This study provides a general good method for researchers interested in the identification and quantification of uridine modifications in various biological samples.