A simple and robust LC-MS method for determination of poly(A) tail length using the Orbitrap Exploris MS systems

Importance of the Topic

Polyadenylation of mRNA plays a critical role in molecule stability and translational efficiency, directly impacting the quality and performance of mRNA-based therapeutics and vaccines. Accurate measurement of poly(A) tail length is essential for ensuring consistency in in vitro transcription processes, monitoring mRNA integrity, and optimizing delivery formulations in research and industrial settings.

Objectives and Study Overview

This study presents a direct, high-resolution LC-MS method for determining poly(A) tail length in synthetic mRNA using Thermo Scientific Orbitrap Exploris mass spectrometers. Key goals include achieving single-nucleotide resolution, maintaining mass accuracy within 5 ppm, and implementing a streamlined data processing workflow via Chromeleon eWorkflow for rapid reporting and cross-instrument consistency assessment.

Methodology and Instrumentation

Sample Preparation

• Synthetic Cas9 mRNA from IVT was digested with RNase T1 to cleave at guanosine residues, preserving intact poly(A) tails.
• Poly(A) tails were purified using oligo(dT)25 magnetic beads, dried, and reconstituted for analysis.

Chromatography and Mass Spectrometry

• Separation on DNAPac RP column via UHPLC with ion-pairing reagents (15 mM dibutylamine, 25 mM HFIP, and acetonitrile gradient).
• Detection using Orbitrap Exploris 240 and MX mass spectrometers in intact protein mode at 180 000 resolution (m/z 200).
• Data processing with Chromeleon CDS 7.3.2 and Xtract deconvolution for isotopically resolved mass spectra.

Results and Discussion

Chromatographic separation yielded a clean poly(A) tail peak between 5.6 and 5.9 minutes. Deconvoluted spectra revealed monoisotopic masses corresponding to tail lengths of 117–132 adenosines, with a median length near 124–125. Mass errors remained below 5 ppm across three Orbitrap Exploris systems, demonstrating reproducibility and robustness of the method.

Benefits and Practical Applications

This direct LC-MS approach eliminates the need for cDNA conversion or indirect assays, offering single-nucleotide resolution and high mass accuracy. The integrated Chromeleon eWorkflow enables automated reporting, supporting quality control in mRNA therapeutics production, stability studies, and regulatory compliance.

Future Trends and Opportunities

Expanding this methodology to diverse mRNA constructs and high-throughput formats could further accelerate vaccine and therapeutic development. Integration with automated sample handling and advanced bioinformatics will enhance downstream analytics and real-time monitoring of production workflows.

Conclusion

A robust, high-resolution LC-MS method on Orbitrap Exploris platforms provides accurate, reproducible poly(A) tail length measurements with streamlined data processing. This workflow supports critical quality attributes in mRNA-based research and manufacturing.

Instrumentation Used

• Thermo Scientific DNAPac RP column and Vanquish Horizon UHPLC system
• Orbitrap Exploris 240 mass spectrometer
• Orbitrap Exploris MX mass detector
• Chromeleon CDS version 7.3.2 with Xtract deconvolution

References

• Clifford TH et al NPJ Vaccines (2022) 7 1
• Neuzil KM NEJM (2023)
• Qin S et al Signal Transduction and Targeted Therapy (2022) 7 166
• Mulligan MJ et al Nature (2020) 586 589–593
• Beverley M et al Anal Bioanal Chem (2018) 410 1667–1677
• Thermo Fisher Scientific Application Note 001183