3 hydroxypicolinic acid

Captured miRNAs were purified with a Zip Tip C18 column (Millipore, Billerica, MA, USA) according to the manufacturer’s protocol. Purified samples were mixed with an aqueous solution of 3-hydroxypicolinic acid (Bruker Daltonics, Bremen, Germany) at a ratio of 1:1 (v/v) and applied to the target plate. One microliter of the mixture was applied to an MTP AnchorChip 384 target plate (Bruker Daltonics) and air-dried at room temperature. MALDI-TOF-MS analysis was performed with an ultrafleXtreme MALDI-TOF/TOF mass spectrometer (Bruker Daltonics) operated in negative ion and reflectron modes. Spectra were manually acquired using FlexControl software (v.3.3.108.0) (Bruker Daltonics). An analysis of the RNAs following hydrazine treatment revealed that the peaks contained m5C^{25 (link)}. Methylated adenines were determined to be 6 mA by sequential MS analysis following treatment with dimethylsulfate, which preferentially alkylates the N1 of adenines in RNA^{26 (link)}.

Final fractions of both tetramer and monomer purification were analyzed using MALDI-TOF mass spectrometry (MS) with a Bruker rapifleX MALDI-TOF system (Bruker Daltonics) as previously described (58 (link)). A sinapinic acid (trans-3,5-dimethoxy-4-hydroxycinnamic acid, Bruker Daltonics) saturated solution in 33% acetonitrile/67% water containing 0.1% trifluoroacetic acid was prepared. A 3-hydroxypicolinic acid (Bruker Daltonics) saturated solution was also prepared. A matrix solution was prepared with 3:1 volumes of each solution. A 0.5 ml volume of each sample was mixed with 0.5 ml of the matrix solution and air-dried at room temperature on the MALDI plate for analysis.