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

Significance of the Topic

The analysis of synthetic oligonucleotides by MALDI-TOF mass spectrometry offers a fast and reliable alternative to traditional gel electrophoresis and ethidium bromide staining in genotyping workflows. In particular, the ability to operate in negative ion mode reduces alkali adduct formation and streamlines sample preparation. Demonstrating this approach using the common cystic fibrosis mutation Phe508del provides both clinical relevance and an educational model for molecular biology training labs.

Objectives and Study Overview

This study evaluates the performance of the MALDI-8030 dual polarity benchtop linear MALDI-TOF mass spectrometer for genotyping synthetic oligonucleotides corresponding to wild type and Phe508del-mutated CFTR sequences. The goals are to compare positive and negative ion modes, assess the need for sample desalting, and demonstrate clear differentiation of homozygous, heterozygous, and wild type alleles based on mass measurements.

Methodology and Instrumentation

Synthetic oligonucleotides (100 μM) representing the CFTR target sequences (ATCTTTGGTGTT for wild type and ATTGGTGTT for Phe508del mutant) were prepared in ultrapure water. 3-Hydroxypicolinic acid matrix was mixed with ammonium citrate in acetonitrile/water. Samples for positive mode underwent cation exchange desalting with Dowex resin, whereas negative mode analyses used untreated mixtures. The MALDI-8030 instrument operated in linear mode at both polarities, acquiring spectra of [M+H]+ and [M–H]– species.

Main Results and Discussion

Negative ion mode spectra exhibited clean baselines free of sodium and potassium adducts, even without desalting, while positive mode required sample clean-up and still showed minor adduct peaks. The average [M–H]– m/z values of 3656.43 and 2758.85 corresponded precisely to wild type and Phe508del mutant oligonucleotides, respectively. Heterozygous samples produced distinct peaks for both species, enabling clear genotyping of all three allelic states.

Benefits and Practical Applications

• Elimination of desalting steps reduces hands-on time and consumables.
• High mass accuracy allows unambiguous identification of oligonucleotide variants.
• Rapid spectral acquisition supports faster turnaround compared to electrophoretic methods.
• Suitable for teaching laboratories to illustrate molecular diagnostics workflows.
• Potential for routine genotyping in research and quality control settings.

Future Trends and Applications

Integration of MALDI-TOF oligonucleotide analysis into high-throughput platforms may enable multiplexed detection of multiple mutations. Advances in matrix formulations and instrument sensitivity could extend the method to longer or modified oligonucleotides. Combining this approach with automated sample handling may facilitate clinical diagnostics and personalized medicine applications.

Conclusion

The use of negative ion mode on the MALDI-8030 bench-top MALDI-TOF instrument provides a rapid, accurate, and simplified workflow for oligonucleotide genotyping. This method overcomes limitations of salt adducts and labor-intensive gel assays, offering an effective alternative for both educational and routine laboratory environments.

Reference

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