Advances in soft ionization techniques such as MALDI (matrix-assisted laser desorption/ionization) and ESI (electrospray ionization) have greatly expanded the samples and molecule types amenable to analysis by mass spectrometry (MS). Specifically, these techniques enable the analysis of very large biomolecules, often in their original state or at the very least intact, without having to break them up into smaller fragments.
One essential component of such analyses is ion mobility spectrometry (IMS), which separates ionized molecules based on their mobility in a carrier gas. Think of IMS as a type of medium-free gas-phase chromatography that is highly sensitive to charge, molecular mass, and shape.
IMS detects both drastic and subtle molecular behavior, including intermolecular associations and intramolecular events like conformational changes. For example, protein complexes consisting of A, B, C, D subunits may have different isoforms. The isoforms predominated by one type of interactions, say subunit A with subunit D, might migrate differently in the gas phase from the species formed by the association of subunit B with subunit C, or even subunit A with subunit C. “Since these species will migrate differently, ion mobility will tell you what you have,” says Weibin Chen, Ph.D., director for scientific operations at Waters.
Mass spectrometers capable of making ion mobility measurements have traditionally been confined to core MS labs due to their complexity and cost. More recently, benchtop mass spectrometers, like the Waters Vion Mass Spectrometer, are putting the discriminating power of ion mobility measurements into the hands of more and more scientists.
The other component, not essential but highly supportive of high-throughput native MS analysis, is a front-end chromatographic separation, based on size exclusion, developed by Waters, which renders an ESI-unfriendly sample to one that the mass spectrometer readily ionizes.
Subtle differences
IMS probes protein structural characteristics for both complexed and monomeric proteins. For example, state of aggregation, complexation with proteins or other large molecules, conformational changes, and folding are all fair game for this technique.
Protein-protein interactions, including aggregation, are “relatively simple to identify,” adds Chen. As long as these species, originating in solution phase, remain intact in the gas phase under MS conditions, “you can use the measured masses and work out from the stoichiometry species that are associated. Two-X masses, indicative of dimerization, are recognized very easily.”
A similar strategy may be employed for proteins bound to species that are smaller than proteins, for example peptides, oligonucleotides or DNA, cofactors, and metabolites. Detection depends on the ability of the MS to distinguish among associated species that differ incrementally in molecular weight.
For a monomeric protein analyzed by MS, differentiation of its conformation changes is somewhat more challenging, given that species will possess the same molecular weight regardless of folding status. It is possible, however, based on the difference in the charge envelope to extract quite a bit of information regarding a protein’s higher-order structures.
“Denatured proteins tend to be more highly charged, and the more charges they carry, the lower their m/Z ratios and their corresponding charge envelope appears,” Chen explains.
Proteins, despite their very large size, have a limited number of sites that can be accessed for charging under their native, properly folded state. When unfolded, the protein backbone is more exposed to its surrounding molecules and therefore able to carry more charges. Denatured proteins also have a broader charge envelope than natively folded proteins in solution.
Waters scientists at the company's Customer Evaluation and Demonstration Laboratory in Wilmslow, England. The Waters Vion IMS QTof mass spectrometer, pictured, separates proteins and protein complexes based on their shape, mass, and charge, bringing added separation power to biomolecule characterization and confirmation.
“When more charge-related functional groups are exposed, the protein will generally be more highly charged,” Chen says. “M/z, and charge envelope, can therefore be a measure of a protein product’s extent of denaturation.”
Were stoichiometry and counting charges the only challenges, not much more would remain to this story. However, two issues complicate native protein analysis by IMS: the nature of protein-protein (or protein-large molecule) associations, and correlating what is essentially a gas-phase state to a molecule’s solution properties.
“If the association between proteins is hydrophobic, these hydrophobic interactions weaken when solvent molecules are stripped away during electrospray ionization,” Chen explains. “Maintaining them is challenging because, generally, molecules behave very differently when solvated than when not.”
This is certainly true of small molecules, for example hydrocarbons with few “moving parts”, which are swamped by hydrophobic forces in solution but not in the gas phase. Second- and third-order forces predominate with much larger proteins containing multiple physical and chemical features that govern folding, as well as intramolecular and intermolecular interactions.
“For very large molecules and complexes, much of their folding and aggregative behavior is generally believed to be similar in the gas phase to those properties in solution,” Chen says. The correlation holds well, provided that conditions preserve the protein’s tertiary and quaternary structure, enabling direct characterization of large intact protein assemblies even when solvent molecules are no longer present. “Scientists have observed a memory effect, whereby certain properties carry over from native states even though gas-phase conditions are radically different.” Not all behavior will be preserved, however, so the issue reduces to correlating data from the gas phase to the solution phase.
“This requires care,” Chen continues, because not all properties transfer, and since MS analysis itself has the potential to introduce unanticipated conformational changes. “You have to take information from the MS spectra, and link them back, through orthogonal analysis methods, to solution-phase behavior.” The degree to which such correlations are possible determines the validity of the MS results.
Maintaining the status quo
One key to improving intact protein characterization by MS is to prepare the sample in a way that maintains the protein’s critical structural properties, while removing species that might interfere with ionization or detection. This is usually accomplished by replacing typical buffer species (e.g. detergents, inorganic salts), through dialysis, with volatile salts like ammonium acetate or formate. The problem is that dialysis is time-consuming. Waters solved this problem by introducing a size-exclusion chromatography step before introducing the sample to the ESI probe. If native protein analysis by MS is in your future, you should check out this Waters application note.
“Size exclusion chromatography is essentially a buffer exchange technique that utilizes its size-based separation principle,” Chen says. “Protein solutions containing numerous volatile salts and detergents are bad for ESI, particularly when using static spray methods.” SEC exchanges the most common buffer species in protein samples with ammonium salts, which are themselves volatile, and therefore “disappear” during ionization.