Elucidating the Fine Structure of Fructan Isomers in Agave using Cyclic Ion Mobility Mass Spectrometry

Importance of the Topic

The structural complexity of fructans in Agave tequilana presents analytical challenges with direct consequences for plant physiology studies, industrial processing (notably tequila production) and quality control. Isomeric mixtures of fructo-oligosaccharides (FOS) at the same degree of polymerization (DP) differ in linkage topology and branching, which affects biological function and process performance. Ion Mobility Spectrometry (IMS) coupled to Mass Spectrometry (MS) and interpreted using Collision Cross Section (CCS) values provides a route to discriminate such isomers by gas-phase size and shape, enabling deeper structural resolution than MS alone.

Study Objectives and Overview

This study aimed to resolve and assign fine structural isomers of agave-derived fructans using cyclic ion mobility (cIMS) mass spectrometry. Specific goals were to (1) separate isomeric components of a DP4 fraction from Agave tequilana, (2) compare experimentally measured CCS values (CCSe) with theoretical CCS predictions (CCSt) derived from ab initio structural models, and (3) validate assignments using chromatographic retention time and MS/MS, including comparison to the commercial standard Nystose.

Methods

• Sample preparation: Water extraction of fructans from a 6-year-old Agave tequilana stem, followed by size exclusion chromatography (SEC) on BioGel P2 and fraction collection to enrich DP compositions.
• Ion mobility acquisition: Multipass cyclic IMS experiments were performed to increase separation power; initial experiments used six passes, with additional slicing experiments applying 12 further passes to improve baseline resolution.
• Calibration and CCS derivation: Experimental CCS values (CCSe) were obtained using a Major Mix calibration standard and linear fitting of arrival time distributions (ATDs).
• Theoretical CCS generation: Candidate fructan structures were built and optimized using ab initio methods, and theoretical CCS (CCSt) were computed with the Trajectory Method implemented in IMoS v1.10.
• Orthogonal validation: LC-IMS-MS (ACQUITY UPLC with an Atlantis Premier BEH C18 AX column) and MS/MS were used to corroborate assignments and identify a Nystose standard by retention time, fragmentation and CCS agreement.

Used Instrumentation

• SYNAPT HDMS System (Waters Corporation) for initial DP confirmation.
• SELECT SERIES Cyclic IMS System (Waters Corporation) for multipass ion mobility separations and slicing experiments.
• ACQUITY UPLC System with Atlantis Premier BEH C18 AX column (1.7 µm, 2.1 x 150 mm) for LC-IMS-MS coupling.
• BioGel P2 SEC column (Bio-Rad) for fractionation of fructans.
• Software and models: IMoS v1.10 with the Trajectory Method for CCSt calculations; in-house ab initio 3D optimization workflows.

Main Results and Discussion

• Multipass cIMS of the DP4 fraction (m/z 665) produced six separable mobility components after six passes. Additional targeted slicing of grouped peaks and twelve extra passes achieved near baseline separation between subcomponents.
• Combining LC with cIMS increased observed peak capacity: LC-cIMS revealed up to eight distinct components for the DP4 fraction, indicating that direct infusion cIMS can underestimate isomer diversity.
• Comparison of experimental CCSe and predicted CCSt showed close agreement: differences ranged from ~1.0% to 3.0% across six peaks. The commercial Nystose standard matched one of the components with CCSe and CCSt differing by ~1.0%, and retention time plus MS/MS fragmentation supported the structural assignment.
• Peak assignment strategy leveraged the ranking of ATD and predicted CCS: the lowest ATD was matched to the lowest CCSt and trends were propagated to assign structures to the remaining peaks. This pragmatic approach assumes monotonic relationship between experimental ATD ranking and predicted CCS ranking.
• Results demonstrate that integrating theoretical CCS predictions with high-resolution multipass IMS and orthogonal LC/MS validation provides robust evidence for discriminating closely related fructan isomers.

Benefits and Practical Applications

• Analytical: CCS-guided identification enhances isomer discrimination beyond m/z and MS/MS alone, especially for carbohydrate oligomers where fragment spectra can be ambiguous.
• Industrial: Better resolution of fructan isomers informs quality control in tequila production, monitoring of processing impacts, and standardization of raw material properties.
• Biological and agronomic: Resolving specific fructan linkages aids studies on stress tolerance mechanisms and metabolic profiling in agave breeding and cultivation.
• Methodological: Multipass cyclic IMS combined with LC increases peak capacity without requiring extensive chromatographic method development, accelerating routine analysis of complex oligosaccharide mixtures.

Limitations and Considerations

• Assignment confidence depends on the accuracy of theoretical models; structural sampling and conformational flexibility of sugars can influence CCSt estimates.
• Calibration quality and instrumental conditions affect CCSe precision; environmental factors and ion source variability must be controlled.
• Overlap of isomers with similar CCS may still occur; complementary MS/MS and authentic standards remain important for definitive assignments.

Future Trends and Applications

• Expansion to higher DP fructans and branched structures, leveraging improved modelling and larger CCS libraries for carbohydrates.
• Development of community CCS databases for plant oligosaccharides to accelerate identification without needing standards for every isomer.
• Integration of advanced computational workflows (enhanced conformational sampling, machine-learning CCS predictors) to reduce computational cost and improve prediction accuracy.
• Adoption in industrial QA/QC pipelines for agave raw material grading and process monitoring, and for breeding programs targeting stress-resilient varieties through metabolite phenotyping.
• Combining isotopic labelling and tandem IMS/MSn strategies to localize linkage types and branching points within oligomers.

Conclusion

This work demonstrates that cyclic multipass IMS, when combined with LC separation, MS/MS validation and theoretical CCS predictions, is an effective approach to resolve and assign isomeric fructans from Agave tequilana. The agreement between experimental and predicted CCS values—often within a few percent—and orthogonal confirmation with a Nystose standard support the utility of CCS as a structural discriminator in complex carbohydrate mixtures. Wider adoption and methodological refinements have potential to transform analytical workflows for natural product characterization and industrial monitoring.

References

• Coots J., Gandhi V., Onakoya T., Chen X., Larriba-Andaluz C. A parallelized tool to calculate the electrical mobility of charged aerosol nanoparticles and ions in the gas phase. Journal of Aerosol Science, 147, 105570 (2020).
• Instrument and trademark information as reported by the original work: SYNAPT, SELECT SERIES, Cyclic IMS, ACQUITY, Atlantis and BEH are trademarks of Waters Corporation or its affiliates.