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Spectroscopy of Hydrated Ions

contacts: Matt Bush , Jeremy O'Brien, & Jim Prell

Mechanism of action spectroscopy of hydrated ions.Action spectrum of [Ca(H2O)9]2+ and modes calculated for a structure with D3 symmetry. Free-energy changes for ion binding in biological systems are controlled by both metal-ligand contacts formed in the complex and changes solvation structure around the ion. For ions with high charge density, this leads to interactions between proteins and partially hydrated divalent metal ions and between nucleic acids and divalent metal ions that typically have fully preserved water inner shells. The first step in understanding these and related problems is to gain a detailed understanding of water shell structure around such ions. One strategy to elucidate the factors governing water coordination is to build up a solution, one molecule of water at a time. Spectroscopy has been used in such a way to probe such systems with singly charged anions and cations. 

Despite serving critical chemical and structural roles in biology, experimental studies of hydrated, multiply-charged ions have lagged behind those of singly-charged ions due to significant experimental challenges. In these studies, electrospray ionization and Fourier transform ion cyclotron resonance mass spectrometry are coupled with vibrational spectroscopy to investigate doubly-charged, hydrated clusters in the gas phase. With this technique, increasing hydration results in spectroscopic signatures indicating the completion of first and second solvation shells.

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Zwitterion Stability in the Gas Phase

contacts: Matt Bush ,  Jeremy O'Brien, & Jim Prell

Nonzwitterionic amino acid versus zwitterionic amino acid.

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 recently added a tunable infrared laser system to our 3 tesla ion cylcotron resonance mass spectrometer and have begun to use spectroscopy to investigate zwitteiron formation in amino acid clusters.

Schematic diagram of Berkeley 3T ion cyclotron resonance mass spectrometer, tunable laser, and variable temperature ion cell.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 recently 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 (Jockusch, R. A., Price, W. D. & Williams, E. R. J. Am. Chem. Soc. 1999, 103, 9266-9274. web)

Summar of arginine results.

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Schematic representation of FAIMS separation.Electron capture dissociation (ECD) is a widely used tandem mass spectrometry technique used for rapid de novo protein sequencing. In ECD a thermally generated electron recombines with a multiply protonated peptide ion producing a rich product ion distribution with enough cleavage sites to approach full protein sequence coverage. This is surprising because the recombination energy is estimated to be 4 – 7 eV, a relatively small value when compared to the internal energy of a small protein, which is typically on the order of 20 eV or more. The mechanism of ECD is a controversial subject with evidence supporting both ergodic and nonergodic fragmentation. By directly measuring the energy released as a result of electron capture and the amount that is partitioned into internal modes, the mechanism of this process may be elucidated. Our group has pioneered an approach to measure the internal energy deposition as a result of electron capture by nanometer sized hydrated ions. Electron capture results in the loss of water molecules from the cluster ions and allows the energy deposition to be measured. By comparing these experiments with known and calculated water binding energetic, the energy partitioning upon electron capture by hydrated ions can be investigated.

Representative 2D FAIMS spectrum.In addition to providing information about the mechanism of ECD, our nanocalorimetry method has been used to provide valuable insight into condensed-phase electrochemistry. While absolute gas-phase ionization potentials are well known, the corresponding solution phase values have not been measured. Solution-phase half-cell potentials are measured relative to other redox reactions and even the concept of an absolute solution-phase reduction potential remains controversial. Redox reactions that compose the electrochemical scale are universally referenced to the standard hydrogen electrode (SHE) which is arbitrarily assigned a value of 0 V. Using nanocalorimetry, the absolute reduction potentials of gas-phase clusters are measured and used to obtain the corresponding solution-phase values. Thus, nanocalorimetry provides a gas-phase route to establish an absolute electrochemical scale.

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Ion Mobility/FAIMS

contacts: Alex Donald, Ryan Leib & Rachel Sellon

Schematic representation of FAIMS separation. 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.

Representative 2D FAIMS spectrum.

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.

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Surface Sampling and Microfluidics

contact: Mariam ElNaggar

Surface sampling methodology and results.

For problems involving complex mixtures, orthogonal techniques that complement MS detection are advantageous. Microfluidic systems have the advantages of small sample volumes, integrated chemistries, and high reproducibility, making them well suited for sample manipulation, cleanup, and separation. Surface sampling, as described by Van Berkel and coworkers (Anal.Chem. 2002, 74, 6216-6223), has great utility for probing a variety of molecules off of surfaces at atmospheric pressure. The formation of a microjunction at the end of a microfluidic separation channel decouples separations and ESI for independent optimization. Surface sampling, whether on a solid substrate or at the end of a channel, has the potential to open up new applications in clinical analysis.

Biophysics of Macromolecular ESI-FTICR

contact: Harry Sterling

Crystal structure of barnase/barstar complex showing representative sites of double mutant cycles.

In solution, the proximity of two residues predicts whether the effect of removing them both, equals the sum of the effects from removing them singly, or whether they are coupled.

We will apply the same logic to gas phase protein-protein complexes, using the coupling between residue DDG's to produce a distance map of contacts in the interface: a first approximation of the local gas-phase structure and an experimental probe of water effects.

Using mass specrometry to determine solution-phase protein binding constants.

We have alanine-scanned the binding interface of two proteins, barnase and barstar, and have simple enzymological means to see how mutations affect binding energy in solution. The mass spectrum of a solution of competing monomers gives an instant and high-throughput readout of their relative solution affinities for heterodimer formation. (see right)


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Biomolecule Characterization by Tandem Mass Spectrometry (MS/MS)

contacts: Ryan Leib

ECD spectra of "supercharged" proteins.

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 below (red and green bars denote acidic and proline residues, respectively).An ECD spectrum of cyctochrome with high sequence coverage.

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 promising new 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.


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