Oligonucleotide Sequence Analysis Using MALDImini-1 and LabSolutions Insight Biologics Software

Importance of the Topic

Oligonucleotide therapeutics require rapid and reliable analytical methods for molecular weight and sequence confirmation. The MALDImini-1 digital ion trap mass spectrometer in conjunction with LabSolutions Insight Biologics software addresses the growing demand for compact, high-throughput platforms in oligonucleotide research and quality control.

Objectives and Study Overview

This study demonstrates a workflow for swift oligonucleotide sequence analysis using a synthetic 18-mer model (nusinersen analogue). The goal is to deliver both molecular weight determination and base sequence assignment in approximately ten minutes, leveraging the compact MALDI-DIT-MS system and dedicated software.

Methodology and Instrumentation

• Instrument: MALDImini-1 MALDI digital ion trap mass spectrometer
• Software: LabSolutions Insight Biologics
• Sample: 20 pmol/µL 18-mer oligonucleotide with 2'-O-methoxyethyl and methyl modifications and phosphorothioate backbone
• Matrix: 3-hydroxypicolinic acid and 2,4-dihydroxyacetophenone mixture (1:1 in acetonitrile/water with diammonium hydrogen citrate)
• Sample preparation: Matrix/sample mixed 1:1 v/v, 1 µL spotted and air-dried
• Acquisition settings: MW analysis (LP 60–65 V, DV-1 1300 V, DV-2 7000 V, RF delay 25 ms); Sequence analysis (LP 65–75 V, DV-1 1600 V, DV-2 8000 V, RF delay 15–17 ms; scan ranges m/z 650–5000 and 2000–18000)
• Data output: mzML files imported into the software, theoretical sequence entered, and automated identification performed

Key Results and Discussion

• MW analysis: A prominent [M+H]+ peak at m/z ~7127 matched the theoretical mass with minimal background.
• Sequence analysis: Specific cleavage of the phosphodiester backbone yielded clean, singly charged fragment ions that simplify coverage mapping.
• Software integration: Automatic merging of spectra, immediate fragment assignment, and clear visualization of sequence coverage enhance throughput and data confidence.

Benefits and Practical Applications

• Compact footprint suitable for space-limited labs and powered by a standard 100 V supply
• Rapid end-to-end workflow (~10 min) from sample prep to final report
• Streamlined data interpretation through restricted ion species and user-friendly interface
• Ideal for quality control and R&D of therapeutic oligonucleotides

Future Trends and Opportunities

Advances may include expanded analyte scope to longer or heavily modified oligonucleotides, integration with liquid chromatography for complex mixtures, and improved algorithms for real-time data processing.

• Automated high-throughput sample handling and plate automation
• Enhanced software workflows for novel modifications and isomer differentiation
• Coupling with separation techniques to resolve isobaric species
• Applications in mRNA vaccine QA, CRISPR guide RNA verification, and aptamer screening

Conclusion

The MALDImini-1 and LabSolutions Insight Biologics platform offers a fast, reliable, and compact solution for oligonucleotide mass and sequence analysis, fulfilling the needs of modern therapeutic development and quality assurance laboratories.

Reference

• Fukuyama Y., Uchiyama K., Nishikaze T. Application Note 01-00875-EN, Shimadzu Corporation, Apr. 2025
• Shimadzu Application News 01-00594-EN