A Semi-Automated Method for Sequencing Oligonucleotides using ISD and Pseudo-MS3 on a MALDI-Ion Trap-TOF Mass Spectrometer

Importance of the Topic

Quality control of oligonucleotides is critical for confirming sequence integrity, particularly for therapeutic molecules that often carry chemical modifications to enhance stability and resistance to degradation. Traditional MALDI-TOF linear mode can verify intact mass but lacks sufficient mass accuracy to resolve small mass differences between modified residues. In-source decay (ISD) sequencing offers fragment information but benefits from higher mass accuracy and monoisotopic resolution to unambiguously assign modified bases.

Objectives and Overview of the Study

This work presents a semi-automated workflow for sequencing modified RNA oligonucleotides using ISD on a MALDI-Ion Trap-TOF mass spectrometer coupled with pseudo-MS3. The approach leverages high-resolution ISD fragment spectra and Polymer Analysis software, originally developed for copolymer analysis, to assign sequence compositions iteratively and confirm terminal residue order.

Methodology and Instrumentation

Sample Preparation

• Desalting of RNA samples in deionized water using Dowex 50WX8-200 ion-exchange resin.
• Matrix prepared with 3-hydroxypicolinic acid and ammonium citrate.

Mass Spectrometry

• AXIMA Resonance MALDI-Ion Trap-TOF instrument operated in Mid 850 mode (m/z 850–3500) with laser power increased by ~10% for ISD.
• Acquisition of 500–800 profiles (2 shots per profile).

Data Analysis

• Polymer Analysis software matching experimental masses to candidate compositions within ±200 mDa tolerance.
• Iterative sequencing: initial fragment composition determined from low-m/z ISD peaks, followed by stepwise extension with residue limits adjusted per iteration.
• Terminal fragment orientation confirmed by pseudo-MS3 (MS/MS of selected ISD ions).

Instrumentation Used

• AXIMA Resonance MALDI-Ion Trap-TOF mass spectrometer (Shimadzu, UK)
• Dowex 50WX8-200 ion-exchange resin (Sigma)
• 3-Hydroxypicolinic acid and ammonium citrate matrix components (Fluka)

Main Results and Discussion

Three 2′-O-methyl phosphorothioate–modified RNA oligonucleotides (samples 1–3) were analyzed. High-resolution ISD spectra enabled clear monoisotopic fragment assignment. Polymer Analysis software effectively distinguished correct compositions by isotopic pattern scoring. Complete sequences were obtained for samples 1 and 3 with 100% accuracy; sample 2 achieved 80% correct residues, with two central positions ambiguous due to low-abundance mid-sequence fragments. Pseudo-MS3 confirmed the sequence order of 3′ and 5′ termini.

Benefits and Practical Applications of the Method

• Enhanced mass accuracy and resolution improve confidence in fragment assignments for modified bases.
• Semi-automated data processing accelerates sequence determination and reduces manual interpretation.
• Pseudo-MS3 confirmation provides unambiguous terminal residue order.
• Applicable to quality control in therapeutic oligonucleotide development and industrial analytics.

Future Trends and Applications

Future developments may include automated rule-based filtering to minimize manual validation and software enhancements to extend reliable read length beyond ~20 mers. Integration with advanced data processing platforms or complementary mass spectrometry techniques could enable high-throughput, fully automated sequencing workflows for complex modified oligonucleotides.

Conclusion

The combination of MALDI-Ion Trap-TOF ISD, Polymer Analysis software, and pseudo-MS3 provides an effective semi-automated approach for sequencing modified RNA oligonucleotides. This method offers high mass accuracy, monoisotopic resolution, and terminal confirmation, improving quality control capabilities despite limitations in mid-sequence fragment abundance.