Interrogating the Quaternary Structure of Noncanonical Hemoglobin Complexes by Electrospray Mass Spectrometry and Collision-Induced Dissociation

Abstract

Various activation methods are available for the fragmentation of gaseous protein complexes produced by electrospray ionization (ESI). Such experiments can potentially yield insights into quaternary structure. Collision-induced dissociation (CID) is the most widely used fragmentation technique. Unfortunately, CID of protein complexes is dominated by the ejection of highly charged monomers, a process that does not yield any structural insights. Using hemoglobin (Hb) as a model system, this work examines under what conditions CID generates structurally informative subcomplexes. Native ESI mainly produced tetrameric Hb ions. In addition, “noncanonical” hexameric and octameric complexes were observed. CID of all these species [(αβ)2, (αβ)3, and (αβ)4] predominantly generated highly charged monomers. In addition, we observed hexamer → tetramer + dimer dissociation, implying that hexamers have a tetramer··dimer architecture. Similarly, the observation of octamer → two tetramer dissociation revealed that octamers have a tetramer··tetramer composition. Gas-phase candidate structures of Hb assemblies were produced by molecular dynamics (MD) simulations. Ion mobility spectrometry was used to identify the most likely candidates. Our data reveal that the capability of CID to produce structurally informative subcomplexes depends on the fate of protein–protein interfaces after transfer into the gas phase. Collapse of low affinity interfaces conjoins the corresponding subunits and favors CID via monomer ejection. Structurally informative subcomplexes are formed only if low affinity interfaces do not undergo a major collapse. However, even in these favorable cases CID is still dominated by monomer ejection, requiring careful analysis of the experimental data for the identification of structurally informative subcomplexes.

Surface-induced Dissociation Mass Spectrometry as a Structural Biology Tool

“…Charge partitioning is intimately linked not only to the charge density of the protein ions but also to the method of dissociation. CID is well-known to eject highly charged monomers from a noncovalent protein complex, leaving an (N-1)­mer intact. ,,,,,,− Multiple monomeric subunits can be stripped from a complex through typical collisional activation with target gas (involving multiple low-efficiency collisions), although whether the cleavages occur simultaneously or (more likely) sequentially is unclear. ,− Sequential removal of monomers was suggested by Benesch et al, who observed production of 11mer of TaHSP16.9 12mer at modest CID collision energies with a concurrent disappearance of the 12mer, but at high collision energies the 11mer species decreased in relative abundance concurrent with appearance of a 10mer. It has also been reported that peripheral subunits are preferentially released. ,,, Wang et al found that nonperipheral subunits could be released through secondary dissociation after primary removal of peripheral subunits or directly from charge-reduced or elongated protein complexes .…”

“…The origin of asymmetric charge partitioning in CID yet symmetric charge partitioning in SID is best contrasted as a multistep “heating” activation method (CID) vs a single high-energy jump activation (SID) (Figure ). ,, There is both experimental evidence ,, and molecular dynamics simulations ,− that asymmetric charge partitioning is the result of a monomer unfolding during the multistep CID process. Benesch et al found a correlation between the proportion of charge retained by CID monomers and the monomer surface area as a fraction of the total surface area of the monomer and (N-1)­mer .…”

Atomistic Insights into the Formation of Nonspecific Protein Complexes during Electrospray Ionization

“…Interestingly, there are also instances where nonspecific clustering is beneficial; for example, protein clusters can serve as model systems for benchmarking mass analyzer performance at high m/z and as a testbed for top-down dissociation experiments. 20,56,57,60 Nonspecific clustering is usually attributed to the CRM, where a nanodroplet containing two or more analyte molecules causes these solutes to "stick" to one another as the droplet dries out. In addition to nonspecific protein−protein contacts, these conditions can cause adduction to other nonvolatile species.…”

“…False-positive outcomes arise from ESI-induced nonspecific clustering. This phenomenon can manifest itself as complex formation from monomeric proteins − or the assembly of complexes into higher-order oligomers. ,− There can also be a mix of specific solution binding and nonspecific clustering. , All of these clustering scenarios complicate the interpretation of native ESI-MS data. Various strategies have been proposed for mitigating this problem, ,,− but it is nonetheless challenging to distinguish specific from nonspecific complexes in a mass spectrum.…”

“…Various strategies have been proposed for mitigating this problem, ,,− but it is nonetheless challenging to distinguish specific from nonspecific complexes in a mass spectrum. Interestingly, there are also instances where nonspecific clustering is beneficial; for example, protein clusters can serve as model systems for benchmarking mass analyzer performance at high m / z and as a testbed for top-down dissociation experiments. ,,, …”