Investigating the Structural Compaction of Biomolecules Upon Transition to the Gas-Phase Using ESI-TWIMS-MS

Abstract

Collision cross-section (CCS) measurements obtained from ion mobility spectrometry-mass spectrometry (IMS-MS) analyses often provide useful information concerning a protein’s size and shape and can be complemented by modeling procedures. However, there have been some concerns about the extent to which certain proteins maintain a native-like conformation during the gas-phase analysis, especially proteins with dynamic or extended regions. Here we have measured the CCSs of a range of biomolecules including non-globular proteins and RNAs of different sequence, size, and stability. Using traveling wave IMS-MS, we show that for the proteins studied, the measured CCS deviates significantly from predicted CCS values based upon currently available structures. The results presented indicate that these proteins collapse to different extents varying on their elongated structures upon transition into the gas-phase. Comparing two RNAs of similar mass but different solution structures, we show that these biomolecules may also be susceptible to gas-phase compaction. Together, the results suggest that caution is needed when predicting structural models based on CCS data for RNAs as well as proteins with non-globular folds. Graphical Abstractᅟ Electronic supplementary materialThe online version of this article (doi:10.1007/s13361-017-1689-9) contains supplementary material, which is available to authorized users.

A Mass‐Spectrometry‐Based Modelling Workflow for Accurate Prediction of IgG Antibody Conformations in the Gas Phase

“…[34,35] Thus,w ehypothesise that IgG molecules may experience precollapse prior to transfer into the gas phase. [34,35] Thus,w ehypothesise that IgG molecules may experience precollapse prior to transfer into the gas phase.…”

“…[29] Native MS mainly uses electrospray ionisation (ESI) for the purpose of creating multiply charged protein ions. Early studies which compared the experimental CCS of antibodies to those calculated from their crystal structures, observed agreater than 30 %discrepancybetween these CCS values, [34,35] thus suggesting collapse of the protein in the gas phase.Such collapse is experienced by nonglobular molecules that are intrinsically flexible or disordered in solution and are capable of conformational change. [31][32][33] TheC RM envisions gradual droplet desolvation leading to production of adry protein ion.…”

“…This is in agreement with an earlier electron microscopy study by Roux et al who observed ad istance of (80 AE 23) between the Fabs and Fc in solution, and an estimated 100-110 for the length of the extended hinge, [6] as well as with other hydrodynamic and solution X-ray scattering studies. Consistent with previous studies of human IgG1-4, [34,35] we observed an approximately 30 % difference between the experimental CCS (CCS exp )a nd model CCS (CCS model ;T able 1), thus suggesting significant structural collapse.D espite the much longer length of IgG3 compared to IgG1, 2, and 4, all IgG antibodies are equally able to collapse,producing aCCS of approximately 7000 2 . Consistent with previous studies of human IgG1-4, [34,35] we observed an approximately 30 % difference between the experimental CCS (CCS exp )a nd model CCS (CCS model ;T able 1), thus suggesting significant structural collapse.D espite the much longer length of IgG3 compared to IgG1, 2, and 4, all IgG antibodies are equally able to collapse,producing aCCS of approximately 7000 2 .…”

Mass spectrometry-enabled structural biology of membrane proteins

“…For example, since hydrophobic interactions are weakened in the gas phase, macromolecular complexes that rely on hydrophobic contacts for assembly may not survive intact during the transfer to the gas phase [82,83]. In addition, the loss of a solvation shell may result in structural reorganization, as evidenced for water soluble proteins, including intrinsically disordered proteins or complexes with 'open' topologies as found in extended polyproteins, such as IgGs or ring-like subunit assemblies [76,81,163]. In these cases it is advisable to perform molecular dynamics simulations to model the behavior of molecules in the gas phase.…”

“…There is much experimental evidence to demonstrate that protein conformations in vacuo are not significantly affected by the ionization process and the loss of the solvent [74,75], although some data suggest that a degree of reorganization does occur [76]. Indeed, there is evidence that structural compaction occurs in unstructured loop regions [77,78], in the case of intrinsically disordered proteins [79,80], and where large cavities may be structural features of proteins/assemblies [81].…”