Oligonucleotide Analysis Using the Compact MALDImini™-1 MALDI Digital Ion Trap Mass Spectrometer

Significance of the Topic

This section highlights the critical role of advanced mass spectrometric methods for the analysis of synthetic oligonucleotides in therapeutic development. Rapid and accurate determination of molecular weight and sequence integrity is essential for ensuring safety and efficacy of oligonucleotide drugs.

Objectives and Study Overview

The goal of this application study is to demonstrate the performance of the compact MALDImini™-1 digital ion trap mass spectrometer in analyzing a synthetic oligonucleotide modelled on the therapeutically relevant sequence mipomersen. Key objectives include precise molecular weight measurement and reliable end-sequence analysis using MSn techniques.

Methodology and Instrumentation

• Sample Preparation: A 20 pmol/μL solution of 20-base synthetic oligonucleotide containing 2′-O-(2-methoxyethyl) and phosphorothioate modifications.
• Matrix System: Equimolar mixture of 3-hydroxypicolinic acid (3-HPA) and 2,4-dihydroxyacetophenone (2,4-DHAP) in acetonitrile/water (50:50, v/v) with 70 mM diammonium hydrogen citrate.
• Deposition: Sample and matrix mixed 1:1 (v/v), 1 μL spotted on target plate, air-dried.
• Acquisition Settings: Laser power, detector voltages (DV-1, DV-2), and RF delay tuned for molecular weight (m/z up to 70,000) and sequence analysis (m/z 650–5000), as detailed in the condition table.

Main Results and Discussion

• Molecular Weight Analysis: The observed [M+H]+ ion at ~7177 Da matched the theoretical mass of the modified oligonucleotide, confirming accurate mass measurement.
• Sequence Analysis: MS1 spectra exhibited clear a/w-type fragment ion ladders. Overlapping peaks near the [M+H]+ precursor were resolved by MS2 analysis of the w5 fragment, enabling precise assignment of terminal bases.
• Combined MS1/MS2 Approach: Integration of MS1 fragment ladders with targeted MS2 spectra allowed comprehensive coverage of the entire sequence, including modification positions.

Benefits and Practical Applications of the Method

• Fast and reliable determination of molecular weight and nucleotide sequence, including chemically modified positions.
• Compact benchtop design conserves laboratory space and facilitates routine QA/QC in research and production environments.
• MSn capabilities enhance confidence in sequence verification, critical for oligonucleotide therapeutic development and quality control.

Future Trends and Potential Applications

Emerging developments may include automation of sample prep, integration of high-throughput screening for oligonucleotide libraries, and advanced MSn workflows for complex modifications. Miniaturization of hardware and AI-driven data interpretation are expected to further expand applications in clinical diagnostics and industrial analytics.

Conclusion

The MALDImini™-1 MALDI-DIT-MS system offers a streamlined, high-performance platform for comprehensive analysis of synthetic oligonucleotides. Its combined capability for accurate mass measurement and multi-stage fragmentation makes it a valuable tool for research laboratories and quality control in oligonucleotide therapeutics.

References

1. McLuckey SA, Journal of the American Society for Mass Spectrometry, 1992, 3, 60–70.