Ion Mobility

Ion mobility-mass spectrometry for structural characterization


About the project

Structurally sensitive techniques such as mass spectrometry (MS), while being an invaluable tool for the structural identification of materials in objects of cultural heritage, can be challenging to employ because of their high sample requirements or the need for front-end methods such as chromatography to separate the protein from complex mixtures. There is an overarching need for new analytical methodologies that improve the information content acquired by MS workflows that do not increase the quantity of precious sample acquired from objects of cultural heritage.  Ion mobility (IM) is a gas-phase technique that separates gas-phase structures based on their collision cross-section (CCS) on the millisecond timescale1. By hyphenating state of the art IM and MS platforms together we can improve the information content associated with the structure of materials in objects of cultural heritage. 

Figure 6 IM-MS Workflow

Ion mobility-mass spectrometry (IM-MS) principles. Ion are generated at the ion source, and allowed to drift through an ion guide filled with neutral gas molecules (e.g. Ar) under the influence of a weak electric field. The ions migrate through the drift tube based on their size, charge, and shape. The ions are then injected into an orthogonal acceleration time-of-flight (oa-TOF) mass analyzer under vacuum for mass-to-charge analysis. A 3-D data set is achieved by the drift time (ms) being contoured to the mass spectra (m/z), with the color scale being scaled to ion abundance.

What is ion mobility?

IM is a technique that separates gas-phase protein structures based on their orientationally averaged collision cross-section (CCS) and has emerged as a useful technique for the characterization of biological macromolecules2. To obtain CCS values, gas-phase protein ions are injected into a drift cell that is filled with an inert gas, e.g. nitrogen or helium.  The ions drift from the entrance to the exit of an ion guide under the influence of a weak electric field. The electric potential forms a gradient that propels the ions towards the exit of the guide. As the ions traverse the guide, they undergo hundreds of thousands of low-energy collisions with the neutral buffer gas. During IM separation, large ions collide more frequently with the buffer gas than those that are smaller, resulting in differences in migration time, or drift time, to the mass spectrometer detector. This drift time can then be converted to a CCS value by using the Mason-Schamp equation, and as such IM separates protein ions in a way that is highly correlated to ion size, shape, and charge (Figure 1)3,4.

When coupled to MS, drift times from IM-MS experiments can be correlated with ion compositions that reveal the influences on protein structure and conformation as a result of sequence changes5,6, small molecule conjugation7, and post-translational modification on proteins as large as antibody structures8,9.

We envision that IM-MS would improve the analysis for works of art in two ways. First, it would allow proteinaceous materials higher order structures to be characterized without the need for material intensive separation techniques typically used to “clean” the sample prior to MS analysis. Secondly, it would simultaneously provide separation of small molecules or dyes which often contain structural isomers that are difficult to separate by traditional MS techniques alone.

Generation of Collisional Induced Unfolding (CIU) Fingerprint. (A) The m/z range for each individual mAb charge states are evaluated for future extraction. (B) The drift time plots associated with each step of activated collision is collected, and unfolding for all charge states can be observed. (C) The drift time distributions are plotted as a function of the collision voltage energy. (D) The CIU stability “fingerprint” is projected as a contour plot with drift time as the y-axis, collision voltage as the y-axis, and ion intensity as the z-axis and representing the color scale. 

Collision induced unfolding

Furthermore, when activating proteins in the gas-phase the resulting unfolded structures observe through such collision induced unfolding (CIU) experiments ,as illustrated in Figure 2, are capable of further differentiating protein structures based on changes to their stabilities associated with domain structure5,10, anion and cation adduction11, ligand and cofactor binding12, as well as post translational modifications such as disulfide bridges and glycosylation patterns13–15. This technique when employed on proteinaceous materials could allow us to generate fingerprints for works of art, and allow us to compare and contrast fingerprints between art works of similar ages, regions, or changes as a results of stress-based degradation. 

Further Reading

(1)      May, J. C.; McLean, J. A. Ion Mobility-Mass Spectrometry: Time-Dispersive Instrumentation. Anal. Chem. 2015, 87 (3), 1422–1436.

(2)      Ben-Nissan, G.; Sharon, M. The Application of Ion-Mobility Mass Spectrometry for Structure/Function Investigation of Protein Complexes. Curr. Opin. Chem. Biol. 2018, 42, 25–33.

(3)      Richardson, K.; Langridge, D.; Dixit, S. M.; Ruotolo, B. T. An Improved Calibration Approach for Traveling Wave Ion Mobility Spectrometry: Robust, High-Precision Collision Cross Sections. Anal. Chem. 2021, 93 (7), 3542–3550.

(4)      Benesch, J. L. P.; Ruotolo, B. T.; Simmons, D. A.; Robinson, C. V. Protein Complexes in the Gas Phase: Technology for Structural Genomics and Proteomics. Chem. Rev. 2007, 107 (8), 3544–3567.

(5)      Watanabe, Y.; Vasiljevic, S.; Allen, J. D.; Seabright, G. E.; Duyvesteyn, H. M. E.; Doores, K. J.; Crispin, M.; Struwe, W. B. Signature of Antibody Domain Exchange by Native Mass Spectrometry and Collision-Induced Unfolding. Anal. Chem. 2018, 90 (12), 7325–7331.

(6)      Hernandez-Alba, O.; Wagner-Rousset, E.; Beck, A.; Cianférani, S. Native Mass Spectrometry, Ion Mobility and Collision Induced Unfolding for Conformational Characterization of IgG4 Monoclonal Antibodies. Anal. Chem. 2018, 15, 8865–8872.

(7)      Debaene, F.; Bœuf, A.; Wagner-Rousset, E.; Colas, O.; Ayoub, D.; Corvaïa, N.; Van Dorsselaer, A.; Beck, A.; Cianférani, S. Innovative Native MS Methodologies for Antibody Drug Conjugate Characterization: High Resolution Native MS and IM-MS for Average DAR and DAR Distribution Assessment. Anal. Chem. 2014, 86 (21), 10674–10683.

(8)      Thompson, N. J.; Rosati, S.; Rose, R. J.; Heck, A. J. R. The Impact of Mass Spectrometry on the Study of Intact Antibodies: From Post-Translational Modifications to Structural Analysis. Chem. Commun. 2013, 49 (6), 538–548.

(9)      Zhang, H.; Cui, W.; Gross, M. L. Mass Spectrometry for the Biophysical Characterization of Therapeutic Monoclonal Antibodies. FEBS Lett. 2014, 588 (2), 308–317.

(10)    Zhong, Y.; Han, L.; Ruotolo, B. T. Collisional and Coulombic Unfolding of Gas-Phase Proteins: High Correlation to Their Domain Structures in Solution. Angew. Chemie Int. Ed. 2014, 53 (35).

(11)    Han, L.; Hyung, S. J.; Ruotolo, B. T. Bound Cations Significantly Stabilize the Structure of Multiprotein Complexes in the Gas Phase. Angew. Chemie – Int. Ed. 2012, 51 (23), 5692–5695.

(12)    Hopper, J. T. S.; Oldham, N. J. Collision Induced Unfolding of Protein Ions in the Gas Phase Studied by Ion Mobility-Mass Spectrometry: The Effect of Ligand Binding on Conformational Stability. J. Am. Soc. Mass Spectrom. 2009, 20 (10), 1851–1858.

(13)    Tian, Y.; Han, L.; Buckner, A. C.; Ruotolo, B. T. Collision Induced Unfolding of Intact Antibodies: Rapid Characterization of Disulfide Bonding Patterns, Glycosylation, and Structures. Anal. Chem. 2015, 87 (22), 11509–11515.

(14)    Wagner, N. D.; Russell, D. H. Defining Noncovalent Ubiquitin Homodimer Interfacial Interactions through Comparisons with Covalently Linked Diubiquitin. J. Am. Chem. Soc. 2016, 138 (51), 16588–16591.

(15)    Jovcevski, B.; Kelly, M. A.; Aquilina, J. A.; Benesch, J. L. P.; Ecroyd, H. Evaluating the Effect of Phosphorylation on the Structure and Dynamics of Hsp27 Dimers by Means of Ion Mobility Mass Spectrometry. Anal. Chem. 2017, 89 (24), 13275–13282.

Research performed in collaboration with…

This page is based upon work supported by the National Science Foundation Mathematical and Physical Sciences divisions ASCEND program under award number CHE-2138107.

Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.