Native Top-Down Mass Spectrometry with Collisionally Activated Dissociation Yields Higher-Order Structure Information for Protein Complexes

Abstract

Native mass spectrometry (MS) of proteins and protein assemblies reveals size and binding stoichiometry, but elucidating structures to understand their function is more challenging. Native top-down MS (nTDMS), i.e., fragmentation of the gas-phase protein, is conventionally used to derive sequence information, locate post-translational modifications (PTMs), and pinpoint ligand binding sites. nTDMS also endeavors to dissociate covalent bonds in a conformation-sensitive manner, such that information about higher-order structure can be inferred from the fragmentation pattern. However, the activation/dissociation method used can greatly affect the resulting information on protein higher-order structure. Methods such as electron capture/transfer dissociation (ECD and ETD, or ExD) and ultraviolet photodissociation (UVPD) can produce product ions that are sensitive to structural features of protein complexes. For multi-subunit complexes, a long-held belief is that collisionally activated dissociation (CAD) induces unfolding and release of a subunit, and thus is not useful for higher-order structure characterization. Here we show not only that sequence information can be obtained directly from CAD of native protein complexes but that the fragmentation pattern can deliver higher-order structural information about their gas- and solution-phase structures. Moreover, CAD-generated internal fragments (i.e., fragments containing neither N-/C-termini) reveal structural aspects of protein complexes.

MASH Native: A Unified Solution for Native Top-Down Proteomics Data Processing

“…The presence of intrachain disulfide bonds limits the fragmentation efficiency of the ADC and reduces sequence coverage by terminal fragment assignment. MASH Native incorporates searching and assignment of internal fragment ions, increasing sequence coverage and revealing sequence coverage of regions bounded by disulfide bonds (Figure S9) to provide additional higher-order structural information for proteins and complexes (Lantz et al, 2021(Lantz et al, , 2022.…”

The Structure of Cyclic Neuropeptide Somatostatin and Octapeptide Octreotide in the Presence of Copper Ions: Insights from Transition Metal Ion FRET and Native Ion Mobility-Mass Spectrometry

“…Native mass spectrometry (MS) has emerged as a sensitive biophysical technique to study biomolecular ions, which are believed to preserve their non-covalent interactions and resemble their native folded state following desolvation. , Various fragmentation methods can be applied to provide structural information of protein–protein, protein–ligand, and protein–metal complexes. , Coupling native MS with ion mobility spectrometry enables conformational studies. , In ion mobility-mass spectrometry (IM-MS), ions with different shapes and charge can be separated based on their mobility in an inert gas region under low electric field conditions. Acquired drift times can be converted into collisional cross sections (CCS) by external calibration and thus provide information on the average size of biomolecular ions. , Förster resonance energy transfer (FRET) measurements are based on a radiation-free energy transfer from a donor to an acceptor fluorophore, which is highly distance dependent. − Combining native MS and FRET has emerged as a useful approach to measure intramolecular distances of mass-selected biomolecular ions. − Furthermore, transition metal ion FRET (tmFRET), a recent expansion of gas-phase FRET, , utilizes metal ions (e.g., copper, nickel, or cobalt) for fluorescence quenching to probe shorter distances (10–40 Å) compared to traditional FRET (20–100 Å).…”

“…9,10 Various fragmentation methods can be applied to provide structural information of protein−protein, protein−ligand, and protein−metal complexes. 11,12 Coupling native MS with ion mobility spectrometry enables conformational studies. 13,14 In ion mobility-mass spectrometry (IM-MS), ions with different shapes and charge can be separated based on their mobility in an inert gas region under low electric field conditions.…”

Fragment Ion Abundance Reveals Information about Structure and Charge Localization in Highly Charged Proteins

“…Although it might be reasonable to expect that the heating process preceding dissociation by collisional activation might erase any memory of solution phase structure, recent results obtained by the Loo lab suggest otherwise. 36 They were able to use higher-energy collisional dissociation (HCD) to obtain structural information about protein complexes under native conditions. Collisional activation can also be coupled with radical-directed dissociation (RDD) to explore protein structure.…”

“…Dissociation based on electron capture or transfer (ECD/ETD) yields high sequence coverage and does not require heating of the ion to initiate dissociation. − Ultraviolet photodissociation (UVPD) is also useful for protein characterization, yielding high sequence coverage through a variety of dissociation mechanisms (including some that heat the protein). − Collisional activation can also be used with intact proteins by way of many low energy collisions (as occurs in an ion trap) or by fewer but higher energy collisions (as occurs in beam-type arrangements). Although it might be reasonable to expect that the heating process preceding dissociation by collisional activation might erase any memory of solution phase structure, recent results obtained by the Loo lab suggest otherwise . They were able to use higher-energy collisional dissociation (HCD) to obtain structural information about protein complexes under native conditions.…”