Separation of phosphorothioated oligonucleotide diastereomers using multiplexed drift tube ion mobility mass spectrometry

Hydrophilic interaction liquid chromatography (HILIC) coupled to drift tube ion mobility spectrometry (DTIMS) was used to separate diastereomers of five-unit oligonucleotides containing 0, 1, 2 or 3 phosphorothioate (PS) linkages. Multiplexed DTIMS (where ions are pulsed into the drift tube according to a pre-encoded sequence) and post-acquisition processing using an innovative demultiplexing tool were investigated. The electric field inside the drift tube was optimized to achieve the highest resolving power. The entrance voltage providing the best two-peak resolution was -1000V with 3-bit multiplexing. Under optimized conditions, the eight diastereomers of an oligonucleotide with three PS linkages (5'-TC∗G∗T∗G-3') could be separated unambiguously. Indeed, those diastereomers differed in their collision cross section (CCS) values. The minimal CCS values difference between two adjacent diastereomers was 0.9% with maximal RSD on CCS values of 0.3%. The use of multiplexed ion mobility and the novel high-resolution demultiplexing tool represents a real breakthrough for resolution enhancement of diastereomers in linear DTIMS.