Enabling Protein and Oligonucleotide Ion Mobility Data Analysis in BioConfirm with PNNL PreProcessor Data Conversions

Significance of Topic

This work addresses a critical bottleneck in biomolecular mass spectrometry: the integration of ion mobility (IM) data into conventional data-dependent acquisition (DDA) workflows. By enabling seamless analysis of proteins and oligonucleotides with an added IM dimension, researchers can achieve higher sequence coverage, resolve isomeric species, and improve confidence in structural assignments without extensive software redevelopment.

Study Objectives and Overview

The study demonstrates a new data conversion workflow using the PNNL PreProcessor to transform all-ions IM/MS data from Agilent instruments into standard LC-MS DDA files compatible with BioConfirm 12.1. Two workflows are presented: one that retains full chromatographic separation and one that collapses the LC axis to focus on IM separation.

Methodology and Instrumentation

Experiments were conducted using:

• Agilent 6560 IM-QTOF with All Ions fragmentation mode
• Agilent 1290 Infinity II LC for chromatographic separations
• Trypsin-digested BSA MS Standard (New England BioLabs) for protein analysis
• Single-stranded 21mer DNA oligonucleotide (Integrated DNA Technologies) for nucleic acid analysis
• PNNL PreProcessor software for IM-to-DDA data conversion
• Agilent IM-MS Browser for feature finding and BioConfirm 12.1 for sequence confirmation

Main Results and Discussion

Oligonucleotide Analysis:

• The 21mer DNA eluted as a single LC peak but yielded multiple charge-state separations in the IM dimension.
• Workflow 2 preserved IM drift time, enabling extraction of drift-time EICs for charge states –3 to –11.
• BioConfirm sequence confirmation achieved high fragment sampling across all charge states.

Protein Analysis:

• BSA digest analyzed by both conventional quad-isolation DDA and converted all-ions IM/MS-to-DDA.
• IM-enabled DDA increased sequence coverage from 67.1% to 78.6% by including singly charged peptides.
• Average identification scores improved with the IM-enabled workflow.
• Comparison of workflows showed that separately processing IM isomers (Workflow 2) yields distinct fragmentation spectra, whereas Workflow 1 merges them.

Benefits and Practical Applications

This approach allows existing DDA-based software to exploit ion mobility without modification, providing:

• Enhanced sequence coverage for proteins and oligonucleotides
• Improved handling of low-abundance and singly charged species
• Resolution of IM isomers for more accurate downstream analysis

Future Trends and Potential Applications

Advances may include automated integration of IM features into proteomics and genomics pipelines, machine-learning-driven isomer classification, and real-time IM-aware acquisition schemes. Expanded adoption in quality control, clinical diagnostics, and structural biology is anticipated.

Conclusion

The PNNL PreProcessor conversion successfully bridges the gap between ion mobility separations and standard DDA workflows. By maintaining critical IM information in formats compatible with BioConfirm, this method enhances sequence coverage, resolves isomers, and improves identification confidence without requiring new analysis platforms.

References

• Bilbao A. et al. Journal of Proteome Research 2022, 21(3), 798–807.