Hydrophilic Interaction Chromatography and tandem mass spectrometry for the characterization of methylated oligoribonucleotides

Chemical modifications on oligonucleotides, notably N-6-methyladenosine (m 6 A) are known for their impact on diverse classes of RNA including micro (mi)RNAs. However, the characterization of these modifications on intact sequences remains challenging. An ion-pair free hydrophilic interaction liquid chromatography-high resolution tandem mass spectrometry (HILIC-HRMS/MS) workflow was developed for the intact analysis of miRNA analogues within synthesis batches, with length up to 22 nucleotides, including sequences differing by an increase in methylation levels and positional isomers containing the same number of methyl groups. The method combines amide-based HILIC separation to top-down collision-induced dissociation (CID) MS/MS and delivers three levels of characterization in a 20-min single run. It allows (i) the separation and identification of impurities (shortmers) from full length product (FLP), (ii) the chromatographic resolution of global methylation forms (0, 1 and 2 methyl groups) up to 22-mer sequence length and (iii) positional isomers discrimination based on the presence and/or absence of specific fragments. The application of HILIC-MS/MS workflow on complex samples, such as positional isomers with identical m 6 A counts and non-equimolar mixtures of various methylation levels, demonstrated robust identification of each isomer, including in mixed samples and despite differences in isomer concentration. • Complete characterization of methylated oligoribonucleotides up to 22-mer length, by ion-pair-free HILIC-MS/MS. • Three-level characterization achieved in a single 20-min run. • Chromatographic separation according to length and methylation number • Discrimination of positional isomers by diagnostic top-down CID fragments. • Amide-HILIC for analysis of intact methylated microRNA.