Sequence Analysis of Antisense Nucleic Acid Using a MALDI-8030 Benchtop MALDI-TOF Mass Spectrometer

Importance of the Topic

Nucleic acid therapeutics have emerged as a promising pharmaceutical modality due to their ability to target genetic sequences directly. Reliable quality control and sequence verification are critical in drug development and manufacturing. Traditional sequencing via tandem MS and MS/MS can be laborious and complex. The development of matrix-assisted laser desorption ionization in-source decay (MALDI-ISD) on benchtop instruments offers a streamlined alternative for routine sequencing of modified oligonucleotides.

Objectives and Study Overview

This study evaluates the performance of the Shimadzu MALDI-8030 benchtop linear MALDI-TOF mass spectrometer for direct sequencing of antisense oligonucleotides. Three model synthetic antisense nucleic acid therapeutics mipomersen, nusinersen and inotersen were analyzed to demonstrate the feasibility of MALDI-ISD in negative-ion mode.

Methodology and Instrumentation Used

• Sample Preparation: Each oligonucleotide was dissolved to 0.1 mg/mL (13–14 pmol/µL). A 0.5 µL aliquot was mixed on-target with 0.5 µL of matrix solution.
• Matrices: Two MALDI matrices were tested: THAP (2′,4′,6′-Trihydroxyacetophenone monohydrate) and HPA (2-Hydroxypicolinic acid), each at 40 mg/mL in 50 % acetonitrile with 40 mM ammonium citrate dibasic.
• Instrument Settings: Shimadzu MALDI-8030, linear negative-ion mode, laser power tuned for in-source decay, ion gate blanking at 500 µs, external calibration with lock mass at 7175.7 Da.

Main Results and Discussion

• Intact Ions: All three antisense oligonucleotides produced dominant deprotonated molecular ions [M-H]- with minimal cation adduct formation, thanks to ammonium citrate suppression.
• Fragmentation Patterns: MALDI-ISD generated clear a-series ions from the 5′ terminus and w-series ions from the 3′ terminus. These consecutive fragments allowed unambiguous sequence assignment despite chemical modifications such as 2′-MOE and phosphorothioate linkages.
• Matrix Comparison: HPA outperformed THAP for in-source decay fragment ion yield and spectral clarity, enabling more complete sequence coverage.
• Sequencing Capability: The linear TOF configuration of the MALDI-8030, lacking tandem MS capability, still provided sufficient fragmentation information for internal sequence confirmation.

Benefits and Practical Application of the Method

• Simplified Workflow: Direct in-source decay sequencing eliminates the need for complex MS/MS interpretation and collision-induced dissociation.
• Cost and Time Efficiency: Use of a compact benchtop instrument reduces operational costs and accelerates analysis.
• Applicability: Suitable for routine quality control of synthetic oligonucleotides and rapid screening of HPLC-isolated impurities.

Future Trends and Opportunities for Application

• Broader Oligonucleotide Varieties: Extension to other modified therapeutic modalities such as siRNA or mRNA fragments.
• Automated Workflows: Integration into high-throughput screening and automation platforms for drug discovery pipelines.
• Advanced Matrices and Instrumentation: Development of tailored matrices and ionization strategies to enhance fragmentation efficiency and sensitivity.
• Quantitative Sequencing: Coupling MALDI-ISD with label-free quantitation for comprehensive characterization of oligonucleotide therapeutics.

Conclusion

MALDI-ISD on the Shimadzu MALDI-8030 benchtop MALDI-TOF mass spectrometer enables straightforward and reliable sequencing of antisense oligonucleotides without requiring tandem MS instrumentation. The approach provides clear fragmentation patterns, simplifies quality control workflows, and supports the expanding field of nucleic acid therapeutics.

Reference

Shimadzu Corporation. Sequence Analysis of Antisense Nucleic Acid Using a MALDI-8030 Benchtop MALDI-TOF Mass Spectrometer. Application News, First Edition, December 2022.