Surface-Induced Dissociation Mass Spectra as a Tool for Distinguishing Different Structural Forms of Gas-Phase Multimeric Protein Complexes

Abstract

One attractive feature of ion mobility mass spectrometry (IM-MS) lies in its ability to provide experimental collision cross section (CCS) measurements, which can be used to distinguish different conformations that a protein complex may adopt during its gas-phase unfolding. However, CCS values alone give no detailed information on subunit structure within the complex. Consequently, structural characterization typically requires molecular modeling, which can have uncertainties without experimental support. One method of obtaining direct experimental evidence on the structures of these intermediates is utilizing gas-phase activation techniques that can effectively dissociate the complexes into substructures while preserving the native topological information. The most commonly used activation method, collision-induced dissociation (CID) with low-mass target gases, typically leads to unfolding of monomers of a protein complex. Here, we describe a method that couples IM-MS and surface-induced dissociation (SID) to dissociate the source-activated precursors of three model protein complexes: C-reactive protein (CRP), transthyretin (TTR), and concanavalin A (Con A). The results of this study confirm that CID involves the unfolding of the protein complex via several intermediates. More importantly, our experiments also indicate that retention of similar CCS between different intermediates does not guarantee retention of structure. Although CID spectra (at a given collision energy) of source-activated, mass-selected precursors do not distinguish between native-like, collapsed, and expanded forms of a protein complex, dissociation patterns and/or average charge states of monomer products in SID of each of these forms are unique.

Ion Activation Methods for Peptides and Proteins

“…136–137 SID results in formation of CID-like product ions ( b- and y- fragments) of peptides and gives comparable sequence coverage to that observed with CID. In more recent years, it has largely been used to disassemble protein complexes, 138–144 as discussed in a later section; SID has not been adopted for high throughput proteomics.…”

193 nm Ultraviolet Photodissociation Mass Spectrometry of Tetrameric Protein Complexes Provides Insight into Quaternary and Secondary Protein Topology

“…We attributed this to protein unfolding/restructuring occurring in the source and during ion transfer akin to what has been shown with source activation for ECD and SID. 34–36 Source parameters were consequently selected to minimize the possibility of unfolding. UVPD was performed using a single pulse of the laser, the energy of which was varied from 0.5 mJ to 3.0 mJ.…”

Ion Mobility Collision Cross Section Compendium

“…In the past decade (2006–2015), the average ion mass was approximately 1000 Da and represents predominately peptide CCS values, however significant efforts were also made for reporting CCS values of lower mass ions centered around 400 Da, the latter representing analytical interests in short-chain carbohydrates, 142–147 metabolites, 70,125,148–151 and drug-like molecules. 84,87,89,152 Figure 6B contains the distribution of CCS reporting with analyte masses extending up to the megaDalton range, which illustrates the recent analytical trend of utilizing quantitative ion mobility methods to study the structure of large protein assemblies, 50,124,153–159 some of which are annotated in the figure. These studies specifically target IM-based measurements towards the interpretation of molecular structure.…”