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1. Two-Color IRMPD Applied to Conformationally Complex Ions: Probing Cold Ion Structure and Hot Ion Unfolding

   https://doi.org/10.1021/acs.jpca.1c08388  

   Harrilal, Christopher P.; DeBlase, Andrew F.; McLuckey, Scott A.; Zwier, Timothy S. (October 2021, The Journal of Physical Chemistry A)
2. Ion Mobility-Mass Spectrometry and Collision-Induced Unfolding of Designed Bispecific Antibody Therapeutics

   https://doi.org/10.1021/acs.analchem.3c00344  

   Villafuerte-Vega, Rosendo C; Li, Henry W; Slaney, Thomas R; Chennamsetty, Naresh; Chen, Guodong; Tao, Li; Ruotolo, Brandon T (May 2023, Analytical Chemistry)

   Bispecific antibodies (bsAbs) represent a critically important class of emerging therapeutics capable of targeting two different antigens simultaneously. As such, bsAbs have been developed as effective treatment agents for diseases that remain challenging for conventional monoclonal antibody (mAb) therapeutics to access. Despite these advantages, bsAbs are intricate molecules, requiring both the appropriate engineering and pairing of heavy and light chains derived from separate parent mAbs. Current analytical tools for tracking the bsAb construction process have demonstrated a limited ability to robustly probe the higher-order structure (HOS) of bsAbs. Native ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) have proven to be useful tools in probing the HOS of mAb therapeutics. In this report, we describe a series of detailed and quantitative IM-MS and CIU data sets that reveal HOS details associated with a knob-into-hole (KiH) bsAb model system and its corresponding parent mAbs. We find that quantitative analysis of CIU data indicates that global KiH bsAb stability occupies an intermediate space between the stabilities recorded for its parent mAbs. Furthermore, our CIU data identify the hole-containing half of the KiH bsAb construct to be the least stable, thus driving much of the overall stability of the KiH bsAb. An analysis of both intact bsAb and enzymatic fragments allows us to associate the first and second CIU transitions observed for the intact KiH bsAb to the unfolding Fab and Fc domains, respectively. This result is likely general for CIU data collected for low charge state mAb ions and is supported by data acquired for deglycosylated KiH bsAb and mAb constructs, each of which indicates greater destabilization of the second CIU transition observed in our data. When integrated, our CIU analysis allows us to link changes in the first CIU transition primarily to the Fab region of the hole-containing halfmer, while the second CIU transition is likely strongly connected to the Fc region of the knob-containing halfmer. Taken together, our results provide an unprecedented road map for evaluating the domain-level stabilities and HOS of both KiH bsAb and mAb constructs using CIU. 

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3. Metal Ion Binding Induces Local Protein Unfolding and Destabilizes Human Carbonic Anhydrase II

   https://doi.org/10.1021/acs.inorgchem.1c03271  

   McConnell, Kayla D.; Fitzkee, Nicholas C.; Emerson, Joseph P. (January 2022, Inorganic Chemistry)
4. Tools for unbinned unfolding

   https://doi.org/10.1088/1748-0221/20/05/P05034  

   Milton, Ryan; Mikuni, Vinicius; Lee, Trevin; Arratia, Miguel; Wamorkar, Tanvi; Nachman, Benjamin (May 2025, Journal of Instrumentation)

   Machine learning has enabled differential cross section measurements that are not discretized. Going beyond the traditional histogram-based paradigm, these unbinned unfolding methods are rapidly being integrated into experimental workflows. In order to enable widespread adaptation and standardization, we develop methods, benchmarks, and software for unbinned unfolding. For methodology, we demonstrate the utility of boosted decision trees for unfolding with a relatively small number of high-level features. This complements state-of-the-art deep learning models capable of unfolding the full phase space. To benchmark unbinned unfolding methods, we develop an extension of existing dataset to include acceptance effects, a necessary challenge for real measurements. Additionally, we directly compare binned and unbinned methods using discretized inputs for the latter in order to control for the binning itself. Lastly, we have assembled two software packages for the OmniFold unbinned unfolding method that should serve as the starting point for any future analyses using this technique. One package is based on the widely-used RooUnfold framework and the other is a standalone package available through the Python Package Index (PyPI). 

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5. Unraveling Ribonucleic Acid Unfolding: A Quantitative Comparison of Solution and Gas-Phase Unfolding

   https://doi.org/10.1021/acs.analchem.5c01148  

   Anders, Anna G; Anthenien, Jacqueline; Kilde, Ingrid R; Koutmos, Markos; Ruotolo, Brandon T (June 2025, Analytical Chemistry)

   Ribonucleic acids (RNAs) are challenging structural biology targets, as numerous barriers exist to determining their high-resolution structures and specific biological functions. Previous results have highlighted the utility of collision-induced unfolding (CIU) to relatively rapidly assess noncoding (nc)RNA higher-order structure (HOS) information. Yet, there remain many gaps in our understanding of how these data can be related to the structures adopted by RNAs in solution as current correlations are largely qualitative. In this study, we describe significant advancements in RNA CIU. Previous RNA CIU reports reveal minimal-to-no RNA unfolding events (or features) upon being subjected to standard CIU conditions. Here, we increase the RNA CIU information through supercharging and quantitatively evaluate the improved RNA CIU data obtained to solution-phase unfolding data collected across a range of Mg2+ concentrations. Finally, we apply our supercharged CIU experiment to mitochondrial encephalopathy, lactic acidosis, and stroke-like episode (MELAS)-associated mt-tRNA leucine (Leu, UUR) (mt-tRNALeu(UUR)) species. Our data demonstrate strong quantitative correlations between gas-phase and solution-phase RNA unfolding events as a function of Mg2+ and MELAS-associated mutations. Taken together, these results indicate strong, solution-relevant relationships for CIU data collected for these RNAs. We conclude our work by discussing future work targeting RNA CIU annotation, broader biophysical characterization of disease-associated RNAs using CIU, and CIU-enabled transcriptomic analysis. 

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