High-Resolution Ion Mobility Mass Spectrometry for Separation of Oligonucleotide Phosphorothioate Diastereomers

Phosphorothioate (PS) modifications are important chemical modifications across oligonucleotide therapeutics. However, the introduction of PS linkages significantly increases analytical complexity due to the generation of diastereomers. Here, we extend beyond the limited studies of PS diastereomer ion mobility mass spectrometry (IM-MS) through the comprehensive and systematic analysis of model poly-dT PS oligonucleotides using structures for lossless ion manipulation (SLIM) high-resolution IM (HRIM) coupled to high-resolution mass spectrometry (HRMS). For the first time, we thoroughly investigate the impact of charge state, adduct state, oligonucleotide length, sequence composition/symmetry, and the number and location of PS modifications on HRIM-MS separation. Our data showcase that increased sequence asymmetry and lower oligonucleotide m / z ions notably enhance the resolution of mobility separations. Accordingly, we achieve exceptional performance, up to 5.0 peak-to-peak resolution (R pp ) across 5–35 nt PS oligonucleotides, with consistent baseline separation under optimized conditions achieved for each oligonucleotide length. We also uncover critical insights into adduct state selection and highlight scenarios in which the adduct state can potentially lead to misinterpretation of PS diastereomer data. Finally, we demonstrate the application of these findings and methods to resolve diastereomers of a critical digest fragment from a therapeutic single guide RNA (sgRNA) sample. By providing insights into the structural and methodological factors that dictate PS diastereomer separations, the findings from this study provide the most detailed framework to date for optimizing IM-MS analysis of PS-modified oligonucleotides.