Williams Group

Gas-phase Ion Chemistry
at UC Berkeley

Williams Group

Less active projects

Zwitterion Stability in the Gas Phase

All naturally occurring amino acids are nonzwitterionic when isolated in the gas phase, despite existing as zwitterions in aqueous solutions over a wide pH range.  There are many ways to stabilize the zwitterionic form of amino acids in the gas phase, including increasing the proton affinity of the proton accepting group and by forming interactions with molecules and ions.  We have previously studied some aspects of these effects with black body infrared radiative dissociation (BIRD).

We have added a tunable infrared laser system to our 2.7 tesla ion cylcotron resonance mass spectrometer and have begun to use spectroscopy to investigate zwitteiron formation in amino acid clusters. The coupling of infrared spectroscopy, electrospray ionization, and ion cyclotron resonance mass spectrometry offers flexibility (plethora of accessible ion clusters), sensitivity (ions are detected with high S/N), and specificity (ions of interest are isolated). Additionally, ions in these experiments are radiatively equilibrated with their surroundings. Changing the temperature of the ion cell and vacuum chamber enables fine control of the internal energy of these clusters. This enables experiments that probe temperature dependent structures.

We have reported infrared photodissociation spectra of cationized arginine, which has the highest proton affinity of the naturally occurring amino acids.  These spectra show that sodiated and potasiated arginine ions are zwitterionic, whereas protonated and lithiated arginine ions are nonzwitterionic.  In these zwitterionic forms, the side chain is protonated, rather than the N-terminal amino group. These spectra enable specific structural asignments and indicate that larger alkali metals preferentially stabilize the zwitterionic form of arginine, consistent with calculations and previously reported framentation patterns 

Zwitterions and the Role of Solvent

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. 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.

Ion Mobility/FAIMS

We have been exploring the gas phase conformations of proteins and polymers by combining high-field asymmetric waveform ion mobility spectrometry (FAIMS) with FT/ICR mass spectrometry. We have investigated the gas-phase conformations of ubiquitin with this method and have used hydrogen/deuterium (H/D) exchange as additional conformational probe in the gas phase. We found that H/D exchange is orthogonal to the FAIMS separation. Our results demonstrated that many more gas-phase conformations of ubiquitin exist than previously measured.

We then probed these ubiquitin conformations with electron capture dissociation (ECD). Our ECD results have demonstrated that electron capture efficiencies do correlate with the cross section of the ion. The correlation is somewhat counter intuitive in that the more compact conformers, those with a smaller cross section, have the greater electron capture efficiency.

We have also used synthetic polymer systems to better understand the fundamental properties of the FAIMS separation.

Biomolecule Characterization by Tandem Mass Spectrometry (MS/MS)

In MS/MS, an analyte ("precursor") ion is fragmented in the gas phase and the masses of the resulting fragment ions are measured, allowing the structure of the analyte to be deduced. For example, MS/MS of protein and peptide ions can be used to rapidly obtain sequence and post-translational modification information. We are investigating the factors that influence the products formed by MS/MS of protein and peptide ions. Recently we discovered that increasing the charge state of protein ions to high levels produces highly selective backbone cleavage in collisionally activated dissociation (CAD), in which precursor ions are fragmented by colliding them with a target gas. For example, increasing the charge state of the protein cytochrome c to 21+ decreases the number of major backbone cleavages to one, with a clustering of cleavages at neighboring residues. The cleavage maps of the 12+, 16+, and 21+ charge states of cytochrome c are shown here (red and green bars denote acidic and proline residues, respectively).This phenomenon is useful because it allows partial sequences, which are useful for identifying proteins from sequence databases, to be obtained with maximum sensitivity because fewer dissociation channels are competitive in higher charge states.

We also are investigating the factors affecting the products of electron capture dissociation (ECD), a powerful method for determining the sequence and locations of post-translational modifications of a protein/peptide in a single MS/MS experiment. The knowledge we gain from this work will allow us to improve the capabilities of ESI-MS/MS as a tool for identifying and characterizing biomolecules with high speed, sensitivity, and selectivity.

Dissociation Methods

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.

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 (left). The infrared laser beam is guided through a transparent ZnSe window into the ion cell and intersects the ions themselves. If the ion absorbs enough photons it dissociates into 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.

Mechanisms of Charge Partitioning

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.

Fundamentals of Electrospray Ionization

The advent of electrospray ionization (ESI) in the 1980s, recognized by the 2002 Nobel Prize in Chemistry, triggered a sea change in the field of mass spectrometry (MS) by making possible the production of molecular ions of very large 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.

Additionally, we have 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 have also 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.

Noncovalent Interactions

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.

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.