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

Significance of the Topic

Oligonucleotide therapeutics represent a growing class of drugs that target disease mechanisms at the genetic level. Precise molecular characterization of these compounds is critical for their development, quality control, and regulatory approval. Mass spectrometry techniques capable of providing both exact mass measurements and sequence confirmation are therefore essential tools in oligonucleotide analysis.

Objectives and Study Overview

This study evaluates the combined use of a high-resolution ESI-QTOF mass spectrometer (LCMS-9030) and a benchtop linear MALDI-TOF instrument (MALDI-8030) for comprehensive analysis of two phosphorothioated oligonucleotides. Goals include intact mass determination, elemental formula confirmation, and sequence verification via in-source decay (ISD).

Methodology and Instrumentation

Two oligonucleotides with distinct sugar modifications (LNA and phosphorothioate DNA) were prepared at 10 pmol/μL in milliQ water.
Instrumentation and conditions:

• LCMS-9030 ESI-QTOF: negative ion mode, m/z range 500–3000, solvent of 50 mM HFIP, 10 mM DIPEA in acetonitrile at 0.2 mL/min; spectral deconvolution using ReSpect in LabSolutions Insight.
• MALDI-8030: dual polarity linear MALDI-TOF in negative ion ISD mode; matrix of 3-hydroxypicolinic acid with ammonium citrate; rapid transition from MS to ISD by laser intensity adjustment.

Main Results and Discussion

Exact mass measurements obtained by LCMS-9030 showed observed masses of m/z 6711.6733 and 6431.7241 for the LNA and S-oligonucleotides, respectively, with deviations of 0.03 ppm and 0.02 ppm from theoretical values. This confirms elemental formulas with high confidence.
The MALDI-8030 ISD spectra produced extensive fragment ion series (a- and w-ions). For the S-oligo, nearly complete a-ion coverage was achieved except for two 5′-terminal units. The LNA-oligo displayed comprehensive internal sequence information via a-ions, demonstrating ISD’s utility where conventional MS/MS is limited.

Benefits and Practical Applications

The complementary use of ESI-QTOF and MALDI-ISD provides:

• Accurate intact mass and elemental composition confirmation.
• Sequence verification through extensive fragment coverage.
• A streamlined workflow for quality control and characterization of oligonucleotide therapeutics.

Future Trends and Potential Applications

Advances in instrument sensitivity and data analysis software are expected to further enhance throughput and coverage of oligonucleotide sequencing. Integration with automated sample handling and bioinformatic pipelines will facilitate high-throughput screening in drug discovery and manufacturing environments. Emerging MALDI techniques and novel matrices may improve fragmentation efficiency for complex modifications.

Conclusion

The combined analytical platform of LCMS-9030 ESI-QTOF and MALDI-8030 ISD offers a robust solution for detailed molecular characterization of oligonucleotide therapeutics, enabling both precise mass confirmation and reliable sequence information that are challenging with single-technique approaches.

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.