Morpholino antisense oligonucleotides analyses using a compact matrix-assisted laser-desorption/ionization digital-ion-trap mass spectrometer (MALDI-DIT-MS)

Morpholino Antisense Oligonucleotides Analysis by MALDI-DIT-MS

Importance of Morpholino Antisense Oligonucleotide Analysis

As the field of oligonucleotide therapeutics expands, fast and accurate methods to characterize morpholino antisense oligonucleotides are critical for quality control, regulatory compliance and development of novel drugs. Morpholino backbones resist standard collision-induced dissociation, making alternative mass spectrometric techniques especially valuable.

Study Objectives and Overview

This work aimed to demonstrate a compact MALDI-DIT-MS platform for mass measurement and sequence analysis of morpholino antisense oligonucleotides. Model compounds viltolarsen and eteplirsen, representative therapeutic morpholinos, were selected.

Methodology and Experimental Approach

The MALDImini-1 digital ion trap mass spectrometer was employed. Analytes were prepared at 20 pmol/µL in water and mixed 1:1 with a matrix of 3-HPA/THAP at 40 mg/mL in acetonitrile/water (50/50) containing 70 mM ammonium citrate. 1 µL deposits were dried on the MALDI plate. Instrumental settings optimized for mass (Mode 1) and sequence (Mode 3) analysis, including dynode voltage, detector voltage and RF delay, enabled sensitive detection of [M+H]+ ions and extensive fragmentation.

Used Instrumentation

• MALDImini-1 compact MALDI-DIT mass spectrometer
• 3-hydroxypicolinic acid (3-HPA) and 2,4,6-trihydroxyacetophenone (THAP) matrices

Main Results and Discussion

High-sensitivity mass spectra showed clear [M+H]+ peaks for both compounds with minimal in-source fragmentation. Sequence analysis revealed a series of b-ions and x−10 ions spanning the full oligo length, enabling complete sequence coverage. Fragmentation pathways were proposed based on observed cleavage patterns, highlighting unique ion types characteristic of MALDI-DIT-MS.

Benefits and Practical Applications

• Rapid molecular weight confirmation of morpholino oligos
• Full-length sequence mapping including modification sites
• Compact instrument footprint suitable for quality control labs

Future Trends and Prospects

Continued integration of electron-based dissociation with MALDI-DIT may further improve fragmentation uniformity. Miniaturized platforms could facilitate high-throughput screening of therapeutic oligonucleotides. Advances in matrix chemistries may enhance sensitivity and sequence coverage for diverse backbone chemistries.

Conclusion

The compact MALDI-DIT-MS method provided an efficient workflow for both mass determination and comprehensive sequencing of morpholino antisense oligonucleotides. Its ability to generate diagnostic fragment ions paves the way for robust analytical support in oligonucleotide drug development.

References

1) ASMS 2023, ThP571, Fukuyama et al.
2) Anal. Chem. 2023, 95, 16352–16358, Karasawa et al.