Analysis of Oligonucleotide Therapeutics using MALDI-8030 and LCMS-9030

Significance of the Topic

Analytical verification of oligonucleotide therapeutics is becoming increasingly important as these molecules advance in drug development. High-precision mass measurement and reliable sequence confirmation are essential to ensure correct molecular composition, safety, and efficacy of upstream therapeutic agents.

Study Objectives and Overview

This study aimed to combine two complementary mass spectrometry techniques—high-resolution electrospray ionization quadrupole time-of-flight (ESI-QTOF) and in-source decay matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS) —to achieve both exact mass determination and detailed sequence information for oligonucleotides. Two phosphorothioated oligonucleotides, one containing locked nucleic acid (LNA) and the other a standard sugar backbone (S-oligo), were evaluated.

Methodology and Instrumentation

Samples were prepared at 10 pmol/µL in high-purity water. Exact mass measurements were performed on a LCMS-9030 QTOF in negative ion mode using a solvent of 50 mmol/L HFIP, 10 mmol/L DIPEA, and acetonitrile at 0.2 mL/min. Data deconvolution was carried out with ReSpect software in LabSolutions Insight.
MALDI-8030, a dual-polarity linear MALDI-TOF instrument, was used for negative-mode in-source decay (ISD). A matrix solution of 3-hydroxypicolinic acid with ammonium citrate facilitated fragmentation. Transition from molecular ion detection to ISD ladder acquisition was achieved by increasing laser irradiation intensity.

Main Results and Discussion

ESI-QTOF analysis yielded deconvoluted masses of 6711.6733 Da for LNA-oligo (theoretical 6711.6731 Da; 0.03 ppm error) and 6431.7241 Da for S-oligo (theoretical 6431.7240 Da; 0.02 ppm error), confirming elemental formulas.
MALDI-ISD spectra exhibited extensive ladders of w-ions (3′-end fragments) and a-ions (5′-end fragments). The S-oligo produced nearly complete a-series coverage, while the LNA-oligo delivered comprehensive internal sequence information. Minor gaps occurred at termini due to matrix signal overlap.

Benefits and Practical Applications

• Accurate intact mass confirmation to sub-ppm precision.
• Rapid sequence verification via MALDI-ISD without tandem MS.
• Combined workflow strengthens quality control protocols in oligonucleotide manufacturing.

Future Trends and Applications

Advances in MALDI-TOF resolution and automated ISD data interpretation are expected to further streamline sequence mapping. Integration with high-throughput workflows and bioinformatics pipelines will enable routine application in QA/QC labs and biopharmaceutical development.

Conclusion

The complementary use of LCMS-9030 ESI-QTOF and MALDI-8030 ISD provides both ultra-accurate mass measurement and robust sequence information for oligonucleotide therapeutics. This hybrid approach overcomes limitations of conventional MS/MS techniques and offers a powerful platform for molecular characterization.

Reference

1) Shimizu H, Jinno F, Morohashi A, Yamazaki Y, Yamada M, Kondo T, Asahi S. J Mass Spectrom. 2012 Aug;47(8):1015-22.