Negative Mode Analysis of Synthetic Oligonucleotides using the MALDI-8030 Dual Polarity Benchtop MALDI-TOF Mass Spectromet

Importance of the topic

Synthetic oligonucleotides are central tools in molecular biology, diagnostics and therapeutic development. Rapid and reliable genotyping of DNA fragments, such as mutations in the CFTR gene causing cystic fibrosis, is crucial for clinical screening, research and teaching laboratories. Adapting MALDI-TOF mass spectrometry for oligonucleotide analysis offers a faster, less labor‐intensive alternative to traditional gel‐based methods.

Objectives and study overview

This application note evaluates the performance of the MALDI-8030 dual polarity benchtop linear MALDI-TOF mass spectrometer for genotyping synthetic oligonucleotides in negative ion mode. Using the common Phe508del CFTR mutation as a model system, the study compares negative vs positive ion detection, examines adduct formation, and demonstrates the ability to distinguish wild type, homozygous mutant and heterozygous samples.

Methodology and Used Instrumentation

The study employed:

• MALDI-8030 Dual Polarity Benchtop Linear MALDI-TOF (Shimadzu)
• 3-Hydroxypicolinic acid (3-HPA) MALDI matrix with ammonium citrate dibasic
• Dowex® cation exchange resin for desalting in positive mode
• Synthetic oligonucleotides (Merck): ATCTTTGGTGTT (wild type), ATTGGTGTT (Phe508del mutant)

Oligonucleotides were prepared at 100 µM and mixed 1:2 with matrix prior to spotting. Negative mode analyses were performed without desalting, while positive mode samples underwent ion‐exchange cleanup.

Main results and discussion

Negative ion mode spectra displayed clean [M–H]⁻ peaks at m/z 3656.43 (wild type) and m/z 2758.85 (mutant) with excellent mass accuracy. In contrast, positive mode spectra required desalting and still exhibited minor Na⁺/K⁺ adducts. The dual polarity MALDI-8030 resolved all three genotypes: wild type, homozygote and heterozygote, enabling clear mass separation and reliable genotyping without gel electrophoresis.

Benefits and practical applications

• Elimination of desalting steps and adduct interferences
• High throughput screening of PCR products
• Reduced analysis time compared to gel electrophoresis and ethidium bromide detection
• Enhanced teaching laboratory workflows for student training
• Potential integration into routine genotyping laboratories

Future trends and potential applications

Advances in benchtop MALDI-TOF instrumentation and matrix chemistries may further improve sensitivity and resolution for longer or modified oligonucleotides. Multiplexed analyses and automated sample preparation could extend applications to high-volume genotyping, next-generation sequencing fragment analysis, and diagnostic panels for diverse genetic disorders.

Conclusion

The MALDI-8030 in negative ion mode offers a streamlined, accurate and cost‐effective workflow for oligonucleotide genotyping. By avoiding desalting and adduct formation, it provides a robust alternative to gel‐based methods, suitable for both educational and routine clinical research settings.

References

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