Proteins often act in large assemblies in the cellular environment to complete tasks necessary to life. With electrospray ionization,1
it is possible to liberate intact, noncovalent assemblies of biomolecules from their native solution environment into the gas phase. In recent years, great strides have been made in the size of assemblies accessible to analysis by mass spectrometry (MS). Complexes that have molecular masses in excess of 1 MDa, including whole ribosomes2–5
and virus particles,4,5
have been studied using MS. A key advantage of mass spectrometry for the analysis of noncovalent biomolecular complexes is that the stoichiometry of the complex can be determined from a simple mass measurement.6
Once in the gas phase, information about the structures of the biomolecules can be obtained using two-dimensional mass spectrometry, in which an ion of interest is mass selected and reacted and structural information inferred from the resulting mass-analyzed product ions. Information about the sequence of peptides,7
can be obtained from dissociation experiments. Such dissociation methods have also been used to determine the location of posttranslational modifications in proteins.7,12,13
Extensive work has been done to understand fragmentation mechanisms of biomolecules to obtain the maximum amount of structural information from a dissociation spectrum. More subtle probes of structure, such as ion mobility,14–17
gas-phase H/D exchange,18,19
and proton-transfer reactivity,21–23
have been used to obtain information about intramolecular noncovalent interactions in biomolecules. Proteins and peptides can adopt different conformations in the gas phase. Information about the intrinsic structure of biomolecules obtained from these experiments provides insights into the role of water in biomolecule structure.24,25
Limited structural information about noncovalent complexes has also been obtained using two-dimensional mass spectrometry. Several studies have indicated that some specific interactions that occur in solution can remain intact during the process of electrospray ionization.26–29
For example, one study showed that the gas-phase dissociation activation energy of a complimentary DNA duplex is higher than those of the noncomplementary DNA homodimers.26
The gas-phase dissociation activation energies and the solution-phase binding energies of several complementary DNA duplexes were found to be correlated. These and other gas-phase measurements, supported by molecular modeling calculations, indicate that the Watson–Crick base pairing that exists for complementary DNA duplexes in solution can be preserved in the gas phase. Recent work by Gabelica et al. shows that the gas-phase kinetic stability of DNA duplexes as a function of collision energy parallels the calculated solution-phase dissociation enthalpies.27
This observation provides additional support that Watson–Crick base pairing and specific DNA base stacking interactions are preserved in the gas phase. Although some specific interactions that exist in solution can be preserved in the gas phase, this is not always the case.30
Other correlations between gas-phase and solution-phase properties of noncovalent complexes have been reported. For example, myoglobin has a noncovalently bound heme ligand. Dissociation kinetics of apomyoglobin ions formed from solutions of different composition are different.31
To determine the contributions that hydrogen bonds provide in the binding of heme to myoglobin, Douglas and co-workers mutated the amino acids responsible for heme binding and systematically reduced the number of hydrogen bonds in the complex.29
A correlation was observed between the solution-phase Arrhenius activation energy of heme loss for the various mutants and their gas-phase kinetic stabilities. An exception occurs for some mutations of residue 92 that decrease the solution-phase activation energy but increase the gas-phase kinetic stability. These experiments suggest that the same hydrogen bonds responsible for heme binding in solution are responsible for binding in the gas phase. These experiments clearly show that these ions can retain a “memory” of their solution-phase structure and that these structural differences can be probed in gas-phase dissociation experiments.
Many dissociation experiments of whole multimeric noncovalent complexes of multiple proteins have been done with the goal of obtaining structural information from these large complexes. Robinson and co-workers used ESI to form ions of the intact E. coli
50S ribosomal subunit which is a noncovalent complex consisting of 33 proteins and 2 strands of RNA.3
Upon collisional activation of the complex in the gas phase, several identifiable proteins were ejected. These proteins contained a disproportionately high degree of charging for their mass compared to that of the original complex. The authors concluded that it is feasible to begin mapping subunit interactions with mass spectrometry.
A key obstacle to using mass spectrometry for the general analysis of protein complexes is a poor understanding of how these complexes dissociate in the gas phase. Many groups have reported a seemingly odd dissociation behavior for large multimeric protein complexes in which a small subunit, typically a protein monomer, is ejected from the complex, with the monomer carrying away a disproportionate amount of charge for its mass relative to the mass of the remaining complex.32–36,38,39
For example, streptavidin exists in solution as a tetramer in which each straptavidin molecule is identical to the others. Gas-phase dissociation of the 14+ charge state of the tetrameric complex results primarily in the formation of the 7+ monomer and the 7+ trimer.36
No dimer ions are observed. Thus, the monomer, which is only one-fourth of the mass of the overall complex, carries off half of the charge!
To explain such highly asymmetric charge partitioning, Smith and co-workers suggested a liquid drop model in which the protein complex is treated as a sphere.36
Upon activation, a fragment “drop” is produced that has a higher surface area-to-mass ratio than the original complex and thus is able to remove a disproportionate amount of charge. This model could not account for the extent of asymmetry, so they speculated that the dissociation of the tetramer may occur by a Coulombically driven process in which a monomer species becomes “unraveled” and is ejected from the aggregate with a disproportionately large share of the charge.34
Some support for this hypothesis was presented by Felytsyn et al., who measured the blackbody infrared radiative dissociation (BIRD) Arrhenius activation parameters for the dissociation of homopentameric Shiga-like toxin I.32
factors as high as 1039
were reported, indicating an unusually high transition-state entropy consistent with a large structural change taking place during the dissociation process. The authors suggested that the charge enrichment of the leaving subunit is energetically unfavorable, but a transfer of charge destabilizes the complex sufficiently that charge enrichment is entropically favorable. Heck and co-workers studied the dissociation of four protein homodimers and found that, even for such relatively simple complexes, asymmetric charge partitioning can occur.38
Heck and co-workers concluded that the liquid drop model as originally proposed by Smith and co-workers36
could not quantitatively account for this result.38
Here, we show that the charge partitioning between the dissociation products of protein homodimers is a function of several parameters. These include the charge state of the complex, the dissociation energy, the solution composition from which these complexes are formed, and the conformational flexibility of the proteins in the complex. These results are consistent with those of Felitsyn et al., where charge asymmetry in the dissociation products is energetically unfavorable, but is driven by high entropy.32
These results also demonstrate that the conformational flexibility of the proteins in the complex is a key part of the asymmetric charge partitioning process.