Matthew F. Bush

Collision Cross Sections of Proteins and Their Complexes: A Calibration Framework and Database for Gas-Phase Structural Biology

Collision cross sections in both helium and nitrogen gases were measured directly using a drift cell with RF ion confinement inserted within a quadrupole/ion mobility/time-of-flight hybrid mass spectrometer (Waters Synapt HDMS, Manchester, U.K.). Collision cross sections for a large set of denatured peptide, denatured protein, native-like protein, and native-like protein complex ions are reported here, forming a database of collision cross sections that spans over 2 orders of magnitude. The average effective density of the native-like ions is 0.6 g cm(-3), which is significantly lower than that for the solvent-excluded regions of proteins and suggests that these ions can retain significant memory of their solution-phase structures rather than collapse to globular structures. Because the measurements are acquired using an instrument that mimics the geometry of the commercial Synapt HDMS instrument, this database enables the determination of highly accurate collision cross sections from traveling-wave ion mobility data through the use of calibration standards with similar masses and mobilities. Errors in traveling-wave collision cross sections determined for native-like protein complexes calibrated using other native-like protein complexes are significantly less than those calibrated using denatured proteins. This database indicates that collision cross sections in both helium and nitrogen gases can be well-correlated for larger biomolecular ions, but non-correlated differences for smaller ions can be more significant. These results enable the generation of more accurate three-dimensional models of protein and other biomolecular complexes using gas-phase structural biology techniques.

Structural characterization of drug-like compounds by ion mobility mass spectrometry: comparison of theoretical and experimentally derived nitrogen collision cross sections.

We present the use of drug-like molecules as a traveling wave (T-wave) ion mobility (IM) calibration sample set, covering the m/z range of 122.1-609.3, the nitrogen collision cross-section (Ω(N(2))) range of 124.5-254.3 Å(2) and the helium collision cross-section (Ω(He)) range of 63.0-178.8 Å(2). Absolute Ω(N(2)) and Ω(He) values for the drug-like calibrants and two diastereomers were measured using a drift-tube instrument with radio frequency (RF) ion confinement. T-wave drift-times for the protonated diastereomers betamethasone and dexamethasone are reproducibly different. Calibration of these drift-times yields T-wave Ω(N(2)) values of 189.4 and 190.4 Å(2), respectively. These results demonstrate the ability of T-wave IM spectrometry to differentiate diastereomers differing in Ω(N(2)) value by only 1 Å(2), even though the resolution of these IM experiments were ∼40 (Ω/ΔΩ). Demonstrated through density functional theory optimized geometries and ionic electrostatic surface potential analysis, the small but measurable mobility difference between the two diastereomers is mainly due to short-range van der Waals interactions with the neutral buffer gas and not long-range charge-induced dipole interactions. The experimental RF-confining drift-tube and T-wave Ω(N(2)) values were also evaluated using a nitrogen based trajectory method, optimized for T-wave operating temperature and pressures, incorporating additional scaling factors to the Lennard-Jones potentials. Experimental Ω(He) values were also compared to the original and optimized helium based trajectory methods.

Ion mobility mass spectrometry of peptide ions: effects of drift gas and calibration strategies.

One difficulty in using ion mobility (IM) mass spectrometry (MS) to improve the specificity of peptide ion assignments is that IM separations are performed using a range of pressures, gas compositions, temperatures, and modes of separation, which makes it challenging to rapidly extract accurate shape parameters. We report collision cross section values (Ω) in both He and N(2) gases for 113 peptide ions determined directly from drift times measured in a low-pressure, ambient temperature drift cell with radio-frequency (rf) ion confinement. These peptide ions have masses ranging from 231 to 2969 Da, Ω(He) of 89-616 Å(2), and Ω(N(2)) of 151-801 Å(2); thus, they are ideal for calibrating results from proteomics experiments. These results were used to quantify the errors associated with traveling-wave Ω measurements of peptide ions and the errors concomitant with using drift times measured in N(2) gas to estimate Ω(He). More broadly, these results enable the rapid and accurate determination of calibrated Ω for peptide ions, which could be used as an additional parameter to increase the specificity of assignments in proteomics experiments.

Charge-state dependent compaction and dissociation of protein complexes: Insights from ion mobility and molecular dynamics.

Collapse to compact states in the gas phase, with smaller collision cross sections than calculated for their native-like structure, has been reported previously for some protein complexes although not rationalized. Here we combine experimental and theoretical studies to investigate the gas-phase structures of four multimeric protein complexes during collisional activation. Importantly, using ion mobility-mass spectrometry (IM-MS), we find that all four macromolecular complexes retain their native-like topologies at low energy. Upon increasing the collision energy, two of the four complexes adopt a more compact state. This collapse was most noticeable for pentameric serum amyloid P (SAP) which contains a large central cavity. The extent of collapse was found to be highly correlated with charge state, with the surprising observation that the lowest charge states were those which experience the greatest degree of compaction. We compared these experimental results with in vacuo molecular dynamics (MD) simulations of SAP, during which the temperature was increased. Simulations showed that low charge states of SAP exhibited compact states, corresponding to collapse of the ring, while intermediate and high charge states unfolded to more extended structures, maintaining their ring-like topology, as observed experimentally. To simulate the collision-induced dissociation (CID) of different charge states of SAP, we used MS to measure the charge state of the ejected monomer and assigned this charge to one subunit, distributing the residual charges evenly among the remaining four subunits. Under these conditions, MD simulations captured the unfolding and ejection of a single subunit for intermediate charge states of SAP. The highest charge states recapitulated the ejection of compact monomers and dimers, which we observed in CID experiments of high charge states of SAP, accessed by supercharging. This strong correlation between theory and experiment has implications for further studies as well as for understanding the process of CID and for applications to gas-phase structural biology more generally.

Effects of Alkaline Earth Metal Ion Complexation on Amino Acid Zwitterion Stability: Results from Infrared Action Spectroscopy

The structures of isolated alkaline earth metal cationized amino acids are investigated using infrared multiple photon dissociation (IRMPD) spectroscopy and theory. These results indicate that arginine, glutamine, proline, serine, and valine all adopt zwitterionic structures when complexed with divalent barium. The IRMPD spectra for these ions exhibit bands assigned to carboxylate stretching modes, spectral signatures for zwitterionic amino acids, and lack bands attributable to the carbonyl stretch of a carboxylic acid functional group. Structural and spectral assignments are strengthened through comparisons with absorbance spectra calculated for low-energy structures and the IRMPD spectra of analogous ions containing monovalent alkali metals. Many bands are significantly red-shifted from the corresponding bands for amino acids complexed with monovalent metal ions, owing to increased charge transfer to divalent metal ions. The IRMPD spectra of arginine complexed with divalent strontium and barium are very similar and indicate that arginine adopts a zwitterionic form in both ions. Calculations indicate that nonzwitterionic forms of arginine are lowest in free energy in complexes with smaller alkaline earth metal cations and that zwitterionic forms are preferentially stabilized with increasing metal ion size. B3LYP and MP2 calculations indicate that zwitterionic forms of arginine are lowest in free energy for M = Ca, Sr, and Ba.

Defining the mechanism of polymerization in the serpinopathies

The serpinopathies result from the ordered polymerization of mutants of members of the serine proteinase inhibitor (serpin) superfamily. These polymers are retained within the cell of synthesis where they cause a toxic gain of function. The serpinopathies are exemplified by inclusions that form with the common severe Z mutant of α1-antitrypsin that are associated with liver cirrhosis. There is considerable controversy as to the pathway of serpin polymerization and the structure of pathogenic polymers that cause disease. We have used synthetic peptides, limited proteolysis, monoclonal antibodies, and ion mobility-mass spectrometry to characterize the polymerogenic intermediate and pathological polymers formed by Z α1-antitrypsin. Our data are best explained by a model in which polymers form through a single intermediate and with a reactive center loop-β-sheet A linkage. Our data are not compatible with the recent model in which polymers are linked by a β-hairpin of the reactive center loop and strand 5A. Understanding the structure of the serpin polymer is essential for rational drug design strategies that aim to block polymerization and so treat α1-antitrypsin deficiency and the serpinopathies.

Traveling‐wave ion mobility mass spectrometry of protein complexes: accurate calibrated collision cross‐sections of human insulin oligomers

RATIONALE The collision cross-section (Ω) of a protein or protein complex ion can be measured using traveling-wave (T-wave) ion mobility (IM) mass spectrometry (MS) via calibration with compounds of known Ω. The T-wave Ω-values depend strongly on instrument parameters and calibrant selection. Optimization of instrument parameters and calibration standards are crucial for obtaining accurate T-wave Ω-values. METHODS Human insulin and the fast-acting insulin aspart under native-like conditions (ammonium acetate, physiological pH) were analyzed on Waters SYNAPT G1 and G2 HDMS instruments. The calibrated T-wave Ω-values of insulin monomer, dimer, and hexamer ions were measured using many different combinations of denatured and native-like calibrants (masses between 2.85 and 256 kDa) and T-wave conditions. Drift-tube Ω-values were obtained on a modified SYNAPT G1. RESULTS Insulin T-wave Ω-values were measured at 26 combinations of T-wave velocity and amplitude. Optimal sets of calibrants were identified that yield Ω-values with minimal dependence on T-wave conditions and calibration plots with high R(2)-values. The T-wave Ω-values determined under conditions satisfying these criteria had absolute errors <2%. Structural differences between human insulin and fast-acting insulin aspart were probed with IM-MS. Insulin aspart monomers have increased flexibility, while hexamers are more compact than human insulin. CONCLUSIONS Accurate T-wave Ω-values that are indistinguishable from drift-tube values are obtained when using (1) native-like calibrants with masses that closely bracket that of the analyte, (2) T-wave velocities that maximize the R(2) of the calibration plot for those calibrants, and (3) at least three replicates at T-wave velocities that yield calibration plots with high R(2).

SCFFBXL3 ubiquitin ligase targets cryptochromes at their cofactor pocket

The cryptochrome (CRY) flavoproteins act as blue-light receptors in plants and insects, but perform light-independent functions at the core of the mammalian circadian clock. To drive clock oscillations, mammalian CRYs associate with the Period proteins (PERs) and together inhibit the transcription of their own genes. The SCFFBXL3 ubiquitin ligase complex controls this negative feedback loop by promoting CRY ubiquitination and degradation. However, the molecular mechanisms of their interactions and the functional role of flavin adenine dinucleotide (FAD) binding in CRYs remain poorly understood. Here we report crystal structures of mammalian CRY2 in its apo, FAD-bound and FBXL3–SKP1-complexed forms. Distinct from other cryptochromes of known structures, mammalian CRY2 binds FAD dynamically with an open cofactor pocket. Notably, the F-box protein FBXL3 captures CRY2 by simultaneously occupying its FAD-binding pocket with a conserved carboxy-terminal tail and burying its PER-binding interface. This novel F-box-protein–substrate bipartite interaction is susceptible to disruption by both FAD and PERs, suggesting a new avenue for pharmacological targeting of the complex and a multifaceted regulatory mechanism of CRY ubiquitination.

Traveling-wave ion mobility mass spectrometry of protein complexes

RATIONALE The collision cross-section (Ω) of a protein or protein complex ion can be measured using traveling-wave (T-wave) ion mobility (IM) mass spectrometry (MS) via calibration with compounds of known Ω. The T-wave Ω-values depend strongly on instrument parameters and calibrant selection. Optimization of instrument parameters and calibration standards are crucial for obtaining accurate T-wave Ω-values. METHODS Human insulin and the fast-acting insulin aspart under native-like conditions (ammonium acetate, physiological pH) were analyzed on Waters SYNAPT G1 and G2 HDMS instruments. The calibrated T-wave Ω-values of insulin monomer, dimer, and hexamer ions were measured using many different combinations of denatured and native-like calibrants (masses between 2.85 and 256 kDa) and T-wave conditions. Drift-tube Ω-values were obtained on a modified SYNAPT G1. RESULTS Insulin T-wave Ω-values were measured at 26 combinations of T-wave velocity and amplitude. Optimal sets of calibrants were identified that yield Ω-values with minimal dependence on T-wave conditions and calibration plots with high R(2)-values. The T-wave Ω-values determined under conditions satisfying these criteria had absolute errors <2%. Structural differences between human insulin and fast-acting insulin aspart were probed with IM-MS. Insulin aspart monomers have increased flexibility, while hexamers are more compact than human insulin. CONCLUSIONS Accurate T-wave Ω-values that are indistinguishable from drift-tube values are obtained when using (1) native-like calibrants with masses that closely bracket that of the analyte, (2) T-wave velocities that maximize the R(2) of the calibration plot for those calibrants, and (3) at least three replicates at T-wave velocities that yield calibration plots with high R(2).

Absolute Standard Hydrogen Electrode Potential Measured by Reduction of Aqueous Nanodrops in the Gas Phase

In solution, half-cell potentials are measured relative to those of other half cells, thereby establishing a ladder of thermochemical values that are referenced to the standard hydrogen electrode (SHE), which is arbitrarily assigned a value of exactly 0 V. Although there has been considerable interest in, and efforts toward, establishing an absolute electrochemical half-cell potential in solution, there is no general consensus regarding the best approach to obtain this value. Here, ion-electron recombination energies resulting from electron capture by gas-phase nanodrops containing individual [M(NH3)6]3+, M = Ru, Co, Os, Cr, and Ir, and Cu2+ ions are obtained from the number of water molecules that are lost from the reduced precursors. These experimental data combined with nanodrop solvation energies estimated from Born theory and solution-phase entropies estimated from limited experimental data provide absolute reduction energies for these redox couples in bulk aqueous solution. A key advantage of this approach is that solvent effects well past two solvent shells, that are difficult to model accurately, are included in these experimental measurements. By evaluating these data relative to known solution-phase reduction potentials, an absolute value for the SHE of 4.2 +/- 0.4 V versus a free electron is obtained. Although not achieved here, the uncertainty of this method could potentially be reduced to below 0.1 V, making this an attractive method for establishing an absolute electrochemical scale that bridges solution and gas-phase redox chemistry.