Defect formation on surfaces bombarded by energetic multiply charged proteins: Implications for the conformation of gas-phase electrosprayed ions

Indirect information on the conformation of highly charged molecular ions may be obtained by monitoring their collisional cross sections and the course of simple gas-phase reactions such as hydrogen-deuterium exchange. In this work, another indirect but more visually oriented approach is explored: electrosprayed protein ions are accelerated toward a highly oriented pyrolytic graphite surface and the resulting single-ion defects are imaged by scanning force and tunneling microscopy. All protein impacts generated shallow hillocks: the shapes depended on the identity and charge state of the incident protein. Lysozyme and myoglobin, both compact, globular proteins in the native state, produced compact, almost circular hillocks. However, hillocks generated by myoglobin that had been denatured in the solution phase were elongated, and the elongation was positively correlated with the charge state of the ion. It appears that structural information about gas-phase multiply charged proteins can be derived from imprints generated by energetic protein impacts on surfaces.     

Infrared photodissociation of non-covalent adducts of electrosprayed nucleotide ions

Efficient dissociation of gaseous non-covalent adducts of multiply charged DNA anions can be effected by infrared irradiation to yield minimal dissociation of the DNA molecular ions-far less dissociation than by collisional activation. Examples include removal of adducted impurities from 100-mer DNA anions and removal of all 14 adducted molecules of 1,4-diaminobutane from M^15? to M^17? of a 50-mer DNA.     

Electron capture dissociation of electrosprayed protein ions for spatially resolved hydrogen exchange measurements

Mass spectrometry (MS) methods involving gas-phase fragmentation hold considerable promise for analyzing regioselective deuteration patterns of proteins following solution-phase amide hydrogen exchange (HX). However, the general viability of such an approach is questionable due to the possible occurrence of intramolecular hydrogen migration ("scrambling"), which tends to randomize or distort the spatial isotope distribution. Rand et al. (J. Am. Chem. Soc. 2008, 130, 1341-1349) have recently reported the application of electron capture dissociation (ECD) for measuring deuteration patterns of short peptides with very little scrambling by FT-MS. The current work shows that even much larger systems such as the 76-residue protein ubiquitin can be successfully analyzed by ECD following solution-phase HX. The resulting c and z. ion deuteration levels are in remarkable agreement with previous NMR data, demonstrating that the extent of scrambling and/or other gas-phase artifacts is negligible. These results open the door to future experiments on the folding, structure, and dynamics of proteins by HX/ECD-FT-MS.     

Conformer-dependent proton-transfer reactions of ubiquitin ions

The conformations of ubiquitin ions before and after being exposed to proton transfer reagents have been studied by using ion mobility/mass spectrometry techniques. Ions were produced by electrospray ionization and exposed to acetone, acetophenone, n-butylamine, and 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene. Under the conditions employed, the +4 to +13 charge states were formed and a variety of conformations, which we have characterized as compact, partially folded, and elongated, have been observed. The low charge state ions have cross sections that are similar to those calculated for the crystal conformation. High charge states favor unfolded conformations. The ion mobility distributions recorded after ions have been exposed to each base show that the lowest charge state that is formed during proton-transfer reactions favors a compact conformation. More open conformations are observed for the higher charge states that remain after reaction. The results show that for a given charge state, the apparent gas-phase acidities of the different conformations are ordered as compact < partially folded < elongated.     

Infrared photodissociation spectroscopy of electrosprayed ions in a Fourier transform mass spectrometer

Previous gas-phase methods for infrared photodissociation spectroscopy (IRPD) require sample volatility. Our method instead uses electrospray ionization to introduce even large nonvolatile molecules into a Fourier transform mass spectrometer, where extended (>15 s) ion storage makes possible high sensitivity spectral measurements with an OPO laser over a range of 3050-3800 cm(-1). The spectra of 22 gaseous proton-bound amino acid complexes are generally correlated with the H-stretching frequencies established for O-H and N-H functional groups in solution. For theoretical structure predictions of the Gly2H+ and N-acylated Asp2H+ dimers, IRPD spectra clearly differentiate between the predicted lowest energy conformers. In contrast to solution, in the gas phase the glycine zwitterion is approximately 20 kcal/mol less stable than the neutral; however, glycine is clearly zwitterionic in the gaseous GlyLysH+ dimer. The level of theory is inadequate for the larger Lys2H+ dimer, as all low energy predicted structures have free carboxyl O-H groups, in contrast to the IR spectrum. IRPD appears to be a promising new technique for providing unique information on a broad range of biomolecular and other gaseous ions, especially on noncovalent bonding involving O-H and N-H groups.     

Dissociation of different conformations of ubiquitin ions

The fragmentation pathways of different conformations of three charge states of ubiquitin ions are examined using ion mobility/collisional activation/time-of-flight techniques. Mass spectra for fragments for different conformers of a single charge state appear to be identical (within the experimental reproducibility). These results are consistent with a mechanism in which different conformers of each charge state rearrange to similar dissociation transition states prior to fragment formation.     

Amylose-iodine complex formation without KI: Evidence for absence of iodide ions within the complex

Amylose-iodine (AI) complex has been synthesized in aqueous solution without added KI. Complex formation (with solid iodine in amylose solution) is maximized at approximately 35°C and decreases beyond that temperature. Ion-selective electrode (ISE) measurements of an aqueous solution of iodine and AI complex indicate that there is no change in the I⁻ ion concentration when the complex forms. This suggests that I⁻ ions (including I, I, and others) cannot be involved in forming the AI complex. The present work also reports a new and simple method for providing both the iodine-binding capacity (IBC) of amylose and the dissociation mechanism for the AI complex. © 1999 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 37: 2711–2717, 1999     

The role of conformation on electron capture dissociation of ubiquitin

Effects of protein conformation on electron capture dissociation (ECD) were investigated using high-field asymmetric waveform ion mobility spectrometry (FAIMS) and Fourier-transform ion cyclotron resonance mass spectrometry. Under the conditions of these experiments, the electron capture efficiency of ubiquitin 6+ formed from three different solution compositions differs significantly, ranging from 51 +/- 7% for ions formed from an acidified water/methanol solution to 88 +/- 2% for ions formed from a buffered aqueous solution. This result clearly indicates that these protein ions retain a memory of their solution-phase structure and that conformational differences can be probed in an ECD experiment. Multiple conformers for the 7+ and 8+ charge states of ubiquitin were separated using FAIMS. ECD spectra of conformer selected ions of the same charge states differ both in electron capture efficiency and in the fragment ion intensities. Conformers of a given charge state that have smaller collisional cross sections can have either a larger or smaller electron capture efficiency. A greater electron capture efficiency was observed for ubiquitin 6+ that has the same collisional cross section as one ubiquitin 7+ conformer, despite the lower charge state. These results indicate that the shape of the molecule can have a greater effect on electron capture efficiency than either collisional cross section or charge state alone. The cleavage locations of different conformers of a given charge state were the same indicating that the presence of different conformers in the gas phase is not due to difference in where charges are located, but rather reflect conformational differences most likely originating from solution. Small neutral losses observed from the singly- and doubly-reduced ubiquitin 6+ do not show a temperature dependence to their formation, consistent with these ions being formed by nonergodic processes.     

Solvation of electrosprayed ions in the accumulation/collision hexapole of a hybrid Q-FTMS

In an effort to spectroscopically determine the structures of solvated ions composed of nucleic acid bases and amino acids, methods for their gas-phase synthesis have been studied. Ions were electrosprayed and solvated in the accumulation cell of a hybrid Q-FTICR filled with methanol or water vapor at ∼10⁻² bar. There were subsequently transferred to the FTICR cell at 10⁻¹⁰ mbar. Following their isolation in the FTICR, they can be investigated by studying their unimolecular blackbody infrared radiative dissociation (BIRD) or infrared multiple photon dissociation (IRMPD) spectroscopy. The IRMPD spectra for (Ade)₂Li⁺ and (Ade)₂Li(H₂O)⁺ are reported and compared as well as BIRD rate constants for multiply solvated and metalated adenine ions.     

Generic shapes for the conformation analysis of macrocyclic structures

A new algorithm for the systematic generation of conformations of macrocyclic systems is presented. The procedure is based on the concept of generic shapes that are found in such structures. These shapes are characterized by a selection of harmonics which occur in an approximate Fourier representation of the atomic coordinates of the rings. Following a fixed protocol, a limited set of in-plane and out-of-plane circular harmonics is used to define an ensemble of generic ring shapes. These generic shapes are used as start structures for energy minimizations by a given force-field method. To account for the possibility of having several final conformations originating from the same generic shape, the corresponding initial structure is taken several times and subjected to a randomization step before minimization. The resulting conformations that fall within a preset low-energy band are collected and screened for duplicates and enantiomers. The efficiency of this procedure (ratio between the number of accepted conformations and the total number of energy minimizations) depends on the flexibility of the macrocyclic system. The efficiency is generally quite high for very flexible rings. According to the proposed protocol, the number of generic shapes used as start structures grows as the square of N(lnN), where N is the ring size. The algorithm lends itself to conformational analyses of medium-size and large rings as well as of loops spanned between fixed structural units.     

Evidence for beta-chlorocarbenium and beta-bromocarbenium ions

Isotopic perturbation of degenerate equilibrium is used to determine whether tetramethylethylenechloronium and tetramethylethylenebromonium ions are closed 1,2-bridged structures or rapid equilibria of open beta-halocarbenium ions. The observed 13C NMR isotope shifts are consistent with a combination of large equilibrium shifts and small upfield intrinsic shifts. The presence of equilibrium shifts in both halonium ions indicates that these ions are not closed 1,2-bridged structures. Rather, they are best represented by equilibria of beta-halocarbenium ions.     

Ejection of solvated ions from electrosprayed methanol/water nanodroplets studied by molecular dynamics simulations

The ejection of solvated small ions from nanometer-sized droplets plays a central role during electrospray ionization (ESI). Molecular dynamics (MD) simulations can provide insights into the nanodroplet behavior. Earlier MD studies have largely focused on aqueous systems, whereas most practical ESI applications involve the use of organic cosolvents. We conduct simulations on mixed water/methanol droplets that carry excess NH(4)(+) ions. Methanol is found to compromise the H-bonding network, resulting in greatly increased rates of ion ejection and solvent evaporation. Considerable differences in the water and methanol escape rates cause time-dependent changes in droplet composition. Segregation occurs at low methanol concentration, such that layered droplets with a methanol-enriched periphery are formed. This phenomenon will enhance the partitioning of analyte molecules, with possible implications for their ESI efficiencies. Solvated ions are ejected from the tip of surface protrusions. Solvent bridging prior to ion secession is more extensive for methanol/water droplets than for purely aqueous systems. The ejection of solvated NH(4)(+) is visualized as diffusion-mediated escape from a metastable basin. The process involves thermally activated crossing of a ~30 kJ mol(-1) free energy barrier, in close agreement with the predictions of the classical ion evaporation model.     

Pulsed helium introduction into a quadrupole ion trap for reduced collisional quenching during infrared multiphoton dissociation of electrosprayed ions

Previous infrared multiphoton dissociation (IRMPD) experiments utilizing a quadrupole ion trap mass spectrometer yielded limited photodissociation efficiencies. Helium buffer gas continuously infused into the analyzer region at pressures of typically 1 x 10(-3) Torr to improve ion trap performance can collisionally quench photoexcited ions during the IRMPD process. Photodissociation experiments have indicated that uncorrected pressures below 2 x 10(-5) Torr are necessary to avoid collisional deactivation of photoexcited ions. This paper describes IRMPD in the quadrupole ion trap at reduced pressures utilizing a dual-pulsed introduction of helium buffer gas incorporated into the ion trap scan function. The pulsed introduction of helium buffer gas before ion injection allows the efficient trapping of ions injected from an electrospray source and the removal of helium before laser irradiation. A second pulse of helium directly before ion detection improves the intensity of the ion signal. The use of this dual-pulsed inlet of helium for improved IRMPD is demonstrated with the carbohydrate antibiotics neomycin and erythromycin. Copyright 2000 John Wiley & Sons, Ltd.     

Evidence for the role of ions in aerosol nucleation

Aerosol nucleation has been studied experimentally in purified, atmospheric air, containing trace amounts of water vapor, ozone, and sulfur dioxide. The results are compared with model calculations. It is found that an increase in ionization by a factor of 10 increases the production rate of stable clusters by a factor of approximately 3, probably due to ion-induced nucleation.     

Likely conformation types for urethane macrorings and their effects on polyrotaxane formation

Stuart-Briegleb models have been used to consider the possible conformations of urethane macrorings and of polyrotaxanes based on them. It is concluded that a set of conformers from the initial cyclourethanes can exist and that such rings can form ordered clumps during complexing with zinc chloride, where the styrene link can penetrate into the cavity in the macroring, and where the cyclourethane conformation has a considerable effect on the degree of crosslinking by the growing polystyrene chain. The conclusions are in good agreement with experiment.Translated from Teoreticheskaya i Éksperimental'naya Khimiya, Vol. 22, No. 4, pp. 507–511, July–August, 1986.     

Evidence for different types of sites on alumina surface

PASCA has been used to search for evidence on different types of sites existing on alumina surfaces. The results indicate that PtCl ₆ ^2– interacts with different surface sites, forming MSI complexes PtO_xCl_y–S_k, PtO_xCl_y–S_s and PtO_xCl_y–St. Surface sites in different aluminas may be different in chemical and electronic properties.

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. PtCl₆ ^2– , PtO_xCl_y–S_k, PtO_xCl_y–S_S PtO_xCl_y–S_t. .

     

Evidence for a reverse U-shaped conformation of single-chain bolaamphiphiles at the air-water interface

Infrared reflection absorption spectroscopy and X-ray reflectivity have been used to elucidate the molecular orientation and hydrocarbon chain conformation and packing of the symmetric long-chain bolaamphiphiles dotriacontane-1,1'-diyl-bis-[2-(trimethylammonio)ethylphosphate] (PC-C32-PC) and dotriacontane-1,1'-diyl-bis-[2-(dimethylammonio)ethylphosphate] (Me2PE-C32-Me2PE) at the air-water interface. At low surface pressures, these bipolar amphiphiles are found to lie flat on the water surface with a disordered chain. With increasing surface pressure, the alkyl chain becomes more ordered. Concomitantly, the chain is bent pointing into the air, whereas both polar headgroups keep contact with the water subphase. At an area of 0.9-1.1 nm2 per molecule, a surface pressure plateau is reached for both bolaamphiphiles, where the molecules adopt a reverse U-shaped conformation with a strongly tilted alkyl chain. Further compression leads to the formation of 3-D aggregates.