Multi-CRAFTI: Relative Collision Cross Sections from Fourier Transform Ion Cyclotron Resonance Mass Spectrometric Line Width Measurements.

Determination of collision cross sections (CCS) using the cross-sectional areas by the Fourier transform ion cyclotron resonance (CRAFTI) technique is limited by the requirement that accurate pressures in the trapping cell of the mass spectrometer must be known. Experiments must also be performed in the energetic hard-sphere regime such that ions decohere after single collisions with neutrals; this limits application to ions that are not much more massive than the neutrals. To mitigate these problems, we have resonantly excited two (or more) ions of different m/z to the same center-of-mass kinetic energy in a single experiment, subjecting them to identical neutral pressures. We term this approach "multi-CRAFTI". This facilitates measurement of relative CCS without requiring knowledge of the pressure and enables determination of absolute CCS using internal standards. Experiments with tetraalkylammonium ions yield CCS in reasonable agreement with the one-ion-at-a-time CRAFTI approach and with ion mobility spectrometry (IMS) when differences in collision energetics are taken into account (multi-CRAFTI generally yields smaller CCS than does IMS due to the higher collision energies employed in multi-CRAFTI). Comparison of multi-CRAFTI and IMS results with CCS calculated from structures computed at the M06-2X/6-31+G* level of theory using projection approximation or trajectory method values, respectively, indicates that the computed structures have CCS increasingly smaller than the experimental CCS as m/z increases, implying the computational model overestimates interactions between the alkyl arms. For ions that undergo similar collisional decoherence processes, relative CCS reach constant values at lower collision energies than do absolute CCS values, suggesting a means of increasing the accessible upper m/z limit by employing multi-CRAFTI.

Analysis of thrombin-antithrombin complex formation using microchip electrophoresis and mass spectrometry.

Preterm birth (PTB) related health problems take over one million lives each year, and currently, no clinical analysis is available to determine if a fetus is at risk for PTB. Here, we describe the preparation of a key PTB risk biomarker, thrombin-antithrombin (TAT), and characterize it using dot blots, MS, and microchip electrophoresis (µCE). The pH for fluorescently labeling TAT was also optimized using spectrofluorometry and spectrophotometry. The LOD of TAT was measured in µCE. Lastly, TAT was combined with six other PTB risk biomarkers and separated in µCE. The ability to make and characterize TAT is an important step toward the development of an integrated microfluidic diagnostic for PTB risk.

Barriers for Extrusion of a Guest from the Interior Binding Cavity of a Host: Gas Phase Experimental and Computational Results for Ion-Capped Decamethylcucurbit[5]uril Complexes.

Factors affecting the extrusion of guests from metal ion-capped decamethylcucurbit[5]uril (mc5) molecular container complexes are investigated using both collision-induced dissociation techniques and molecular mechanics simulations. For guests without polar bonds, the extrusion barrier increases with increasing guest volume. This is likely because escape of larger guests requires more displacement of the metal ion caps and, thus, more disruption of the ion-dipole interactions between the ion caps and the electronegative rim oxygens of mc5. However, guests larger than the optimum size for encapsulation displace the ion caps prior to collision-induced dissociation, resulting in less stable complexes and lower dissociation thresholds. The extrusion barriers obtained for guests with polar bonds are smaller than those obtained for similarly sized guests without polar bonds. This is likely because the partial charges on the guest allow electrostatic interactions with the ion cap and rim oxygens of mc5 during extrusion, thus stabilizing the extrusion transition state and reducing the extrusion barrier. Results from this study demonstrate simple principles to consider for designing host-guest complexes with specific guest-loss behaviors. Similar trends are observed between the experimental and computational results, demonstrating that molecular mechanics simulations can be used to approximate the relative stability of mc5 molecular container complexes and likely those of other similar complexes.

Quantitative Collision Cross-Sections from FTICR Linewidth Measurements: Improvements in Theory and Experiment.

Two corrections to the equation used in the cross-sectional areas by Fourier transform ion cyclotron resonance ("CRAFTI") technique are identified. In CRAFTI, ion collision cross-sections are obtained from the pressure-dependent ion linewidths in Fourier transform mass spectra. The effects of these corrections on the accuracy of the cross-sections obtained using the CRAFTI technique are evaluated experimentally using the 20 biogenic amino acids and several crown ether complexes with protonated alkyl monoamines. Good absolute agreement is obtained between the CRAFTI cross-sections and the corresponding cross-sections obtained using both static drift ion mobility spectrometry and computational simulations. These results indicate that the CRAFTI cross-sections obtained using the updated equation presented here are quantitatively descriptive of the size and shape of the gas-phase ions. Cross-sections that differ by less than 3% are measured for the isobaric isomers n-butylamine and tert-butylamine complexed with the crown ethers. This level of precision is similar to what has been achieved previously using traveling wave ion mobility devices. These results indicate that CRAFTI can be used to probe subtle structural differences between ions with approximately the same precision as that achieved in traveling wave ion mobility devices. Graphical Abstract ᅟ.

Collisional Cross-Sections with T-Wave Ion Mobility Spectrometry without Experimental Calibration.

A method for relating traveling-wave ion mobility spectrometry (TWIMS) drift times with collisional cross-sections using computational simulations is presented. This method is developed using SIMION modeling of the TWIMS potential wave and equations that describe the velocity of ions in gases induced by electric fields. The accuracy of this method is assessed by comparing the collisional cross-sections of 70 different reference ions obtained using this method with those obtained from static drift tube ion mobility measurements. The cross-sections obtained here with low wave velocities are very similar to those obtained using static drift (average difference = 0.3%) for ions formed from both denaturing and buffered aqueous solutions. In contrast, the cross-sections obtained with high wave velocities are significantly greater, especially for ions formed from buffered aqueous solutions. These higher cross-sections at high wave velocities may result from high-order factors not accounted for in the model presented here or from the protein ions unfolding during TWIMS. Results from this study demonstrate that collisional cross-sections can be obtained from single TWIMS drift time measurements, but that low wave velocities and gentle instrument conditions should be used in order to minimize any uncertainties resulting from high-order effects not accounted for in the present model and from any protein unfolding that might occur. Thus, the method presented here eliminates the need to calibrate TWIMS drift times with collisional cross-sections measured using other ion mobility devices. Graphical Abstract ᅟ.

Microsecond and nanosecond polyproline II helix formation in aqueous nanodrops measured by mass spectrometry.

The 1.5 µs and <400 ns time constants for the formation of polyproline II helix structures in 21 and 16 residue peptides, respectively, are measured using rapid mixing from theta-glass emitters coupled with mass spectrometry. Results from these studies should serve as useful benchmarks for comparison with computational simulation results.

Surface-Induced Protein Unfolding in Submicron Electrospray Emitters.

The charging of protein ions formed by nanoelectrospray ionization (nanoESI) with tips that are between 1.5 µm and 250 nm in outer diameter is compared. More charging is obtained with the smaller tip sizes for proteins that have a net positive charge in solution, and additional high-charge-state distributions are often observed. A single charge-state distribution of holo-myoglobin ions is produced by nanoESI from a slightly acidified aqueous solution with the micron outer diameter tips, but some apo-myoglobin ions are produced with the submicron tips. In contrast, the charge-state distributions for proteins with a net negative charge in solution do not depend on tip size. Both the formation of high charge states and the appearance of higher-charge-state distributions, as well as the loss of the heme group from myoglobin, indicate that a fraction of the protein population is unfolding with the smaller tips. The increased charging with the smaller tip sizes for proteins with a net positive charge but not for proteins with a net negative charge indicates that the unfolding occurs prior to nanoelectrospray ionization as a result of Coulombic attraction between positively charged protein molecules in solution and the glass surfaces of the emitter tips that are negatively charged. These results demonstrate a novel method for producing highly charged protein ions that does not require exposing the proteins to additional chemicals either in solution or in the gas phase.

Electrothermal supercharging of proteins in native MS: effects of protein isoelectric point, buffer, and nanoESI-emitter tip size.

The extent of charging resulting from electrothermal supercharging for protein ions formed from various buffered aqueous solutions using nanoESI emitters with tip diameters between ∼1.5 µm and ∼310 nm is compared. Charging increases with decreasing tip size for proteins that are positively charged in solution but not for proteins that are negatively charged in solution. These results suggest that Coulombic attraction between positively charged protein molecules and the negatively charged glass surfaces in the tips of the emitters causes destabilization and even unfolding of proteins prior to nanoESI. Coulombic attraction to the negatively charged glass surfaces does not occur for negatively charged proteins and the extent of charging with electrothermal supercharging decreases with decreasing tip size. Smaller droplets are formed with smaller tips, and these droplets have shorter lifetimes for protein unfolding with electrothermal supercharging to occur prior to gaseous ion formation. Results from this study demonstrate simple principles to consider in order to optimize the extent of charging obtained with electrothermal supercharging, which should be useful for obtaining more structural information in tandem mass spectrometry.

Ultrafast (1 µs) Mixing and Fast Protein Folding in Nanodrops Monitored by Mass Spectrometry.

The use of theta-glass emitters and mass spectrometry to monitor reactions that occur as fast as one µs is demonstrated. Acidified aqueous solutions containing unfolded proteins are mixed with aqueous ammonium acetate solutions to increase the solution pH and induce protein folding during nanoelectrospray ionization. Protein charge-state distributions show the extent to which folding occurs, and reaction times are obtained from known protein folding time constants. Shorter reaction times are obtained by decreasing the solution flow rate, and reaction times between 1.0 and 22 µs are obtained using flow rates between 48 and 2880 pL/s, respectively. Remarkably similar reaction times are obtained for three different proteins (Trp-cage, myoglobin, and cytochrome c) with folding time constants that differ by more than an order of magnitude (4.1, 7, and 57 µs, respectively), indicating that the reaction times obtained using rapid mixing from theta-glass emitters are independent of protein identity. A folding time constant of 2.2 µs is obtained for the formation of a ß-hairpin structure of renin substrate tetradecapeptide, which is the fastest folding event measured using a rapid mixing technique. The 1.0 µs reaction time obtained here is about an order of magnitude lower than the shortest reaction time probed using a conventional mixer (8 µs). Moreover, this fast reaction time is obtained with a 48 pL/s flow rate, which is 2000-times less than the flow rate required to obtained the 8 µs reaction time using a conventional mixer. These results indicate that rapid mixing with theta-glass emitters can be used to access significantly faster reaction times while consuming substantially less sample than in conventional mixing apparatus.

Investigating protein folding and unfolding in electrospray nanodrops upon rapid mixing using theta-glass emitters.

Theta-glass emitters are used to rapidly mix two solutions to induce either protein folding or unfolding during nanoelectrospray (nanoESI). Mixing acid-denatured myoglobin with an aqueous ammonium acetate solution to increase solution pH results in protein folding during nanoESI. A reaction time and upper limit to the droplet lifetime of 9 ± 2 µs is obtained from the relative abundance of the folded conformer in these rapid mixing experiments compared to that obtained from solutions at equilibrium and a folding time constant of 7 µs. Heme reincorporation does not occur, consistent with the short droplet lifetime and the much longer time constant for this process. Similar mixing experiments with acid-denatured cytochrome c and the resulting folding during nanoESI indicate a reaction time of between 7 and 25 µs depending on the solution composition. The extent of unfolding of holo-myoglobin upon rapid mixing with theta-glass emitters is less than that reported previously ( Fisher et al. Anal. Chem. 2014 , 86 , 4581 - 4588 ), a result that is attributed to the much smaller, ∼1.5 µm, average o.d. tips used here. These results indicate that the time frame during which protein folding or unfolding can occur during nanoESI depends both on the initial droplet size, which can be varied by changing the emitter tip diameter, and on the solution composition. This study demonstrates that protein folding or unfolding processes that occur on the ∼10 µs time scale can be readily investigated using rapid mixing with theta-glass emitters combined with mass spectrometry.

Theta-glass capillaries in electrospray ionization: rapid mixing and short droplet lifetimes.

Double-barrel wire-in-a-capillary electrospray emitters prepared from theta-glass capillaries were used to mix solutions during the electrospray process. The relative flow rate of each barrel was continuously monitored with internal standards. The complexation reaction of 18-crown-6 and K(+), introduced from opposite barrels, reaches equilibrium during the electrospray process, suggesting that complete mixing also occurs. A simplified diffusion model suggests that mixing occurs in less than a millisecond, and contributions of turbulence, estimated from times of coalescing ballistic microdroplets, suggest that complete mixing occurs within a few microseconds. This mixing time is 2 orders of magnitude less than in any mixer previously coupled to a mass spectrometer. The reduction of 2,6-dichloroindophenol by l-ascorbic acid was performed using the theta-glass emitters and monitored using mass spectrometry. On the basis of the rate constant of this reaction in bulk solution, an apparent reaction time of 274 ± 60 µs was obtained. This reaction time is an upper limit to the droplet lifetime because the surface area to volume ratio and the concentration of reagents increase as the droplet evaporates and some product formation occurs in the Taylor cone prior to droplet formation. On the basis of increases in reaction rates measured by others in droplets compared to rates in bulk solution, the true droplet lifetime is likely 1-3 orders of magnitude less than the upper limit, i.e., between 27 µs and 270 ns. The rapid mixing and short droplet lifetime achieved in these experiments should enable the monitoring of many different fast reactions using mass spectrometry.

Effects of cations on protein and peptide charging in electrospray ionization from aqueous solutions.

The effects of eight different cations with ionic radii between 69 and 337 pm on the charging of peptides and proteins with electrospray ionization from aqueous acetate salt solutions are reported. Significant adduction occurs for all cations except NH4(+), and the average protein charge is lower when formed from solutions containing salts compared with solutions without salts added. Circular dichroism and ion mobility results show the protein conformations are different in pure water compared with salt solutions, which likely affects the extent of charging. The average charge of protein and peptide ions formed from solutions with Li(+) and Cs(+), which have Gibbs solvation free energies (GSFEs) that differ by 225 kJ/mol, is similar. Lower charge states are typically formed from solutions with tetramethylammonium and tetraethylammonium that have lower GSFE values. Loss of the larger cations that have the lowest GSFEs is facile when adducted protein ions are collisionally activated, resulting in the formation of lower analyte charge states. This reaction pathway provides a route to produce abundant singly protonated protein ions under native mass spectrometry conditions. The average protein and peptide charge with NH4(+) is nearly the same as that with Rb(+) and K(+), cations with similar GSFE and ionic radii. This indicates that proton transfer from NH4(+) to proteins plays an insignificant role in the extent of protein charging in native mass spectrometry.

Influence of charge repulsion on binding strengths: experimental and computational characterization of mixed alkali metal complexes of decamethylcucurbit[5]uril in the gas phase.

Theory and experiment demonstrate that Coulombic repulsion plays a dominant role in the strength of binding a second cation to a rigid, ditopic host.