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Zwitterions and the Role of Solvent

contacts: Matt Bush

Candidate structure for lithiated valine with six waters

The in vivo structure of biomolecules is the result of both intramolecular interactions intrinsic to the molecule and intermolecular interactions with surrounding molecules and ions. These effects are each significant and often favor radically different structures. For example, amino acids in aqueous solution are zwitterions over a wide pH range, even though nonzwitterionic structures are energetically favored in the gas phase. Clearly, water preferentially stabilizes the zwitterionic form of amino acids. While this general concept is well understood, the full structural impact of water on biomolecular structure remains poorly characterized. Gas-phase studies of biomolecules, such as amino acids and their hydrated clusters, should reveal how water interacts with and influences the structure of such molecules.

Representative mass spectra and kinetics for the loss of one water molecule from lithiated alpha-methyl-proline with one water molecule.

The structures of hydrated, cationized clusters of amino acids arre investigated using blackbody infrared radiative dissociation (BIRD). Briefly, electrospray generated ions are trapped in a Fourier-transform ion cyclotron resonance mass spectrometer. The ion cluster of interest is isolated and undergoes unimolecular dissociation. Kinetics are measured over a wide temperature range and modeled using master equation formalism to determine threshold dissociation energies (Eo) for the loss of a water molecule. Information about structure is deduced by comparing that Eo with those measured from clusters of known structure.


Selected Articles:

Ion Cell Design

Prototype filament cell.EPOC cell currently used in our 9.4 T ion cyclotron resonance mass spectrometer.

Filament Dissociation Test Cell: Enables the dissociation of biomolecules with pseudo-blackbody radiation. (shown left)

Novel Ion Cells: The Extended Pseudo-Open Cylindrical Cell (EPOCC, shown right) enables longer trapping times (> 2400 s), greater ion storage, and increased dynamic range for FT-ICR experiments.

Selected Articles:

Dissociation Methods

BIRD ion activation.

Our group has developed two techniques to obtain dissociation energies for large biomolecules. The blackbody infrared radiative dissociation (BIRD) method has been used to obtain dissociation energies of proteins, oligonucleotides and metal water clusters. These experiments are performed solely in the FT-ICR, because of its ability to trap ions over prolonged periods. In a BIRD experiment, the vacuum chamber of our mass spectrometer is heated using a resistive heating blanket. Blackbody photons emitted from the chamber walls are absorbed by ions which are trapped in the ion cell (left). If rate constants for the dissociation of a particular ion are measured over several temperatures, a dissociation energy can be obtained.

Laser ion activation.

Although BIRD experiments are sufficient for measuring energetics for a wide variety of proteins, nucleic acids, etc., some ions have high dissociation energies. Because of the temperature constraints of our FT-ICR instruments, we have developed a technique to measure dissociation energetics using a CO2 infrared laser to dissociate such ions (right). The infrared laser beam is guided through a transparent ZnSe window into the ion cell and intersects the ions themselves. The ion absorbs enough photons it dissociates into its fragments. Recently, dissociation energies of leucine enkephalin, a small peptide, determined using the CO2 laser agree with that found with BIRD experiments. Laser dissociation methods have an additional advantage in that they are quicker to perform.

In addition to photon activation methods, our lab has investigated the use of collisional activation with a inert background gas. Trapped ions are accelerated with the ion cell, producing energetic collisions. Sustained off resonance irradiation (SORI) experiments have been useful in obtaining dissociation energies.

Selected Articles:

Fundamentals of Electrospray Ionization

Supercharging of proteins during electrospray ionization.

The advent of electrospray ionization (ESI) in the 1980s, recognized by the 2002 Nobel Prize in Chemistry, triggered a quantum leap in the field of mass spectrometry (MS) by making possible the production of molecular ions of very large (?108 daltons) molecules. Using ESI-MS, the molecular masses of proteins, oligonucleotides, synthetic polymers and other large species are measured with unprecedented accuracy. ESI has the additional advantages of being able to be directly interfaced to liquid chromatography and capillary electrophoresis, and producing multiply charged ions. The multiple charging reduces the mass-to-charge (m/z) ratio of large molecular ions to a regime where mass resolving power and mass accuracy excel. Multiply charged ions are also ideal for characterization by tandem mass spectrometry (MS/MS).

We are investigating the mechanism by which gas-phase ions are formed in ESI and the factors contributing to the observed degree of multiple charging and sensitivity. The charging depends on several factors, including analyte size and conformation, competition for charge between analyte and solvent, and instrumental factors.

We recently demonstrated that analyte charging in ESI can be increased to unprecedented levels by adding certain solvents such as m-nitrobenzyl alcohol (m-NBA) into denaturing electrospray solutions ("supercharging"). We also recently demonstrated a direct relationship between the solvent surface tension and the degree of analyte charging observed in ESI-MS. The knowledge we gain from these studies improves our ability to control the charge state of analyte ions formed in ESI. It also expands our understanding of how the ESI process affects the structure of the gas-phase analyte ions.

Selected Articles:

Mechanisms of Charge Partitioning

Charge partitioning in ion fragmentation.

The formation of nonspecific, noncovalent complexes is a phenomenological element of electrospray ionization (ESI). Although the mechanism for the formation of these clusters from ESI is not fully understood, the behavior of clusters as gas-phase ions has been well-studied. Clusters of molecules are often used as models for investigating the properties of matter transitioning from the condensed to the gas phase. While numerous studies of nonspecific, noncovalent cluster formation and dissociation have been conducted using materials as diverse as atomic nuclei, noble gasses, metal clusters, and amino acids, remarkably few studies have probed these issues using biological molecules. By utilizing ESI in conjunction with Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS), we have extended these studies to include cluster dissociation mechanisms of peptides and proteins. We are currently using ESI FT-ICR MS to examine solution and gas-phase complex formation and to study how charge partitioning of nonspecific protein homodimers depends on structure.

Selected Articles:

Noncovalent Interactions

Complementary DNA base pairs.

In electrospray ionization, ions are formed from solutions (aqueous or aqueous/organic) containing the protein or ion of interest. Droplets are formed when a solution is feed through a thin capillary which is kept at a high voltage. Often, protein-protein and DNA-DNA complexes which are known to be present in solution are retained in the gas phase. Evidence for Watson-Crick pairing in the gas phase of complementary bases was recently found for short DNA strands (4-7 mers). Computer Simulations using molecular dynamics (left) show that the bases are still paired after 100 ps at 300K for the A7· T73- ion. Even for single nucleotides, the guanosine-cytosine pair (G· C) is retained in the gas phase.

[Mg(H2O)6]2+

Under the proper conditions, hydrated metal and peptide ions can be formed. By measuring the dissociation energies of successive water losses, information about the structure of these ions can be inferred. The dissociation energies measured for metal ion-(H2O)n clusters are sensitive to the structure of the hydration layers or shells. The illustration on the right is a structure of Mg(H20)62+ with 4 inner shell and 2 outer shell waters obtained from density functional calculations. BIRD dissociation results for Mg(H20)62+ are consistent with the presence of a this (4,2) structure at temperatures > 80 C. At lower temperatures, BIRD results indicate Mg(H20)62+ has all six waters in one solvation shell.

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