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The influence of metal cations and source temperature on the conformations of oxidized insulin chain A (ICA) anions has been investigated in the gas phase by high-resolution ion mobility techniques. Cross sections for non-metalated [ICA-nH]n- (n = 2−6) ions show a distinct Coulomb-driven unfolding transition when four or more protons are removed. Studies of [ICA-6H + M2+]4- ions (where M = Ca, Mn, Co, Ni, Cu, or Zn) show that metal peptide ions favor specific types of conformations that depend upon the binding properties of the metal. Doubly-charged metal ions appear to bind to multiple sites (presumably through interactions with −SO3-, −COO-, and amide groups on various amino acids) and have a pronounced effect on the number and shapes of stable conformations. Generally, [ICA-6H + M2+]4- ions are found to be more compact than their [ICA-4H]4- analogues. Studies of [ICA-5H + Na+]4- ions show that addition of Na+ to the ICA anion has little effect on the peptide structure. The relative stabilities of diffe...

A combinatorial tripeptide library having the general form D-Glu-Xxx-Xxx-CONH2 has been synthesized using a standard mix and split synthetic protocol that is expected to produce 676 components. All components of the mixture were analyzed using a new high-resolution ion mobility/time-of-flight mass spectrometer coupled with an electrospray ionization source. In this approach ions are separated by differences in their gas-phase mobilities prior to being introduced into the mass spectrometer for mass-to-charge analysis. The peptide library includes a wide range of different sequence, structural, and stereo isomers; trends in the number of expected and resolved isomers that are observed at each m/z ratio allow specific synthetic steps that have failed to be identified, even in the presence of other isomers. Information about the relative abundances of different isomers should dramatically improve the reliability of binding affinity studies from direct analysis of mixtures.

The recent development of electrospray ionization (ESI)1 and matrix-assisted laser desorption ionization (MALDI)2 sources for creation of macromolecular ions has brought about a renaissance in the field of mass spectrometry (MS). It is now common to determine molecular weights of large molecules such as proteins to within a few daltons (or less)! MS-based methods for sequencing are proliferating and replacing (or complementing) traditional biochemical methods for many applications. However, analysis of covalent structure is only part of the story. A number of studies have investigated the use of MS strategies for examining noncovalent biomolecular structure. Mounting evidence suggests that some conformational properties of biomolecules in solution are preserved during the ionization process and persist over the transient time that ions exist in mass spectrometers. The persistence of solution-like structure in the gas phase provides opportunities to examine conformation and folding as well as binding interactions (e.g., enzyme-substrate, receptor-ligand interactions) in an entirely new environment. In the past 5 years, a new field involving structural studies of biomolecular ions has emerged. At first glance, the relevance of biomolecular ion structure in the gas phase to studies of functional molecules in solution may seem far removed, perhaps not unlike questions about the relevance of structural measurements of protein crystals 20-30 years ago. Studies of biomolecular ions in the gas phase provide information about the nature of conformation in the absence of solvent; thus, the roles of intramolecular interactions and solvent can be discerned by comparison of similarities between gasand solutionphase structure. Additionally, the utility of MS strategies for sampling noncovalent binding between molecules in solution hinge on a fragile balance of interactions associated with the assembled pair as the ions travel through the mass analyzer. As these strategies become widely used, the differentiation 3037 Chem. Rev. 1999, 99, 3037−3079

High-resolution ion mobility spectrometry has been combined with time-of-flight mass spectrometry for analysis of a combinatorial peptide library that is expected to contain 676 components. In this approach, the components of a mixture of three residue peptides, having the general form (D)Phe-Xxx-Xxx-CONH2 (where Xxx is randomized over 26 residues including 10 naturally occurring amino acids and 16 synthetic forms) were ionized by electrospray ionization. Ion mobility/time-of-flight distributions have been recorded for all ions using a nested drift(flight) time technique. The improvement in resolving power [(t/delta t) = 100-150 for singly charged ions] was illustrated by analysis of a mixture of tryptic digest peptides using high- and low-resolution instruments. The approach allows many components of the library (e.g., structural, sequence, and stereo isomers) that cannot be distinguished by mass spectrometry alone to be resolved. Impurities due to side reactions appear to be minimal, comprising10% of the total ion signal. Direct evidence for approximately 60-70% of the expected peptides is found. Variation in ion abundance for different components indicates that there are differences in solution concentrations or ionization efficiencies for the components.

Collision cross section measurements and molecular modeling calculations have been combined to extract average volumes of amino acid residues in gas-phase peptide ions. The approach uses cross sections for a series of 103 lysine-terminated peptide ions having the general form [XxxnLys+H]+ (where Xxx is any amino acid except Lys, Arg, His, and Cys, and n = 4 to 8). The results show that average volumes of individual Xxx residues in the gas phase are 5 to 15% smaller than when found in protein interiors; the Lys residue is 20% smaller in the gas phase than in protein cores. The relatively small volumes of amino acids in the gas phase compared with protein core volumes are explained by considering that few gaps or crevices along interior regions exist in compact model structures that represent the experimental data. The very small volume of the Lys residue in peptide ions is consistent with the expectation that this amino acid is the charge carrier in most (if not all) of the peptides and is subject to “elec...

High-resolution ion mobility/mass spectrometry methods have been used to examine the influence of solvent composition and capillary temperature on the gas-phase conformations of ubiquitin ions (+6 to +13) formed during electrospray ionization. Three general conformer types are observed: compact forms (favored for the +6 and +7 charge states); partially folded conformers (favored for the +8 and +9 ions); and, unfolded conformers (favored for the +10 to +13 charge states). The populations of different conformers are highly sensitive to solvent composition and capillary temperature used for electrospray ionization. Electrospray of “pseudonative” solutions leads to formation of some conformers that are more compact than those observed for “denatured” solutions. Studies as a function of capillary temperature show that as the capillary temperature is increased, compact and partially folded states undergo unfolding transitions. Compact states from pseudonative solutions unfold at higher temperatures than compact states from denatured solutions. The products of unfolding transitions have similar cross sections for both solution types.

Cross sections for mixtures of tryptic digest peptide ions formed by electrospray ionization have been measured by a new ion mobility/time-of-flight mass spectrometry technique. Analysis of a series of 113 peptides containing 5−10 residues and having a single lysine group located at the C-terminal end show that cross sections are largely dependent upon the amino acid composition of each peptide. Reduced cross sections (which take into account differences in mass) are found to correlate with the fractions of nonpolar or polar aliphatic residues. Average intrinsic contributions to size for individual amino acid residues (referred to as intrinsic size parameters) have been obtained by solving a system of equations that relates the 113 reduced cross sections to the occurrence frequency of each residue within the different sequences. These parameters fall into families according to the physical sizes and chemical properties of the amino acids; contributions to cross section from nonpolar residues are significa...

Injected-ion mobility can be used to characterize conformations, examine protein folding, and complement MS methods.

A method for improving the sensitivity of ion mobility measurements of ions generated from continuous ionization sources such as electrospray ionization is described. The method involves an ion trap interface between the continuous source and the pulsed experiment. The trap allows the electrosprayed ion beam to be accumulated between pulses of the mobility experiment and provides tightly focused (both spatially and temporally) ion pulses for initiating experiments. Using this method, we have improved signal-to-noise ratios by factors of ∼10−30 compared with those of existing approaches and achieved experimental duty cycles of nearly 100%. With these improvements, it is possible to record complex ion mobility distributions in less than 10 s, and we have obtained detection limits of 1.3 pmol for the oligosaccharide maltotetraose. A detailed description of the experimental arrangement is given. An application of the improved methods in which collision-induced fragmentation of biomolecules is monitored by ion...

Injected-ion mobility/mass spectrometry techniques have been used to measure the reduced ion mobilities for negatively charged raffinose, melezitose and α-, β-, and γ-cyclodextrins formed by electrospray ionization. At low injection energies, the mass spectra are dominated by negatively charged (deprotonated) parent ions. At high injection energies, the mass spectra recorded for the cyclodextrins and raffinose display peaks that result from cross-ring cleavage of individual sugar units. Melezitose dissociates by cleavage of the glycosidic bonds. The ion mobility distributions can be used to distinguish between different isomeric forms of parent and fragment ions having the same mass-to-charge ratios.

Ion mobility measurements can be used to obtain structural information for large polyatomic ions in the gas phase. The methods are flexible and can be applied to a wide range of chemical systems. This article reviews the development of these methods and discusses recent applications to complex ions such as atomic clusters and large biomolecules. © John Wiley & Sons, Ltd.

The conformations of gaseous lysozyme ions (+5 through +18) produced by electrospray ionization have been studied in the gas phase using ion mobility mass spectrometry techniques. When solutions containing the disulfide-intact and disulfide-reduced lysozyme are electrosprayed, the gas-phase ions that are produced have distinctly different collision cross sections. Disulfide-intact ions favor two conformer types: a highly folded conformer with a cross section near that calculated for the crystal structure and a partially unfolded conformer that is formed when the ions are injected into the drift tube at high injection voltages. Ions formed from the disulfide-reduced solution have collision cross sections that are much larger than any observed for the disulfide-intact protein, showing that these ions are largely unfolded. Gas-phase proton-transfer reactions in the ion source can be used to favor lower charge states for both solutions. When protons are removed from disulfide-intact lysozyme ions, highly fol...

The conformations of cytochrome c ions (+8 through +18) in the gas phase are examined by simultaneous ion-mobility and hydrogen−deuterium exchange measurements. By varying the voltage used to inject ions into the drift tube it is possible to study H/D exchange of specific conformers observed in the ion-mobility spectra: either very diffuse structures that are favored for all charge states at high injection voltages or compact structures that can be favored for the +8, +9, and +10 charge states at low voltages. The number of exchangeable hydrogens for the diffuse conformer is independent of charge state, with an average value that is quite low: only 63 ± 2 of a possible 198. The compact conformers observed for the +8 through +10 charge states exchange fewer hydrogens (∼46), consistent with the idea that compact structures protect some hydrogens in the gas phase. Many sites that rapidly exchange in solution appear to be restricted for exchange in the gas phase, even for very open conformers.

Ion mobility measurements have been used to examine the geometries of naked BPTI (bovine pancreatic trypsin inhibitor) and cytochrome c ions in the gas phase, as a function of charge. For BPTI, the measured cross sections are close to those estimated for the native solution-phase conformation. Furthermore, gas-phase BPTI retains its compact structure when collisionally heated. These results are consistent with the known stability of BPTI, where the three-dimensional structure is partly locked into place by three covalent disulfide bridges. For cytochrome c, geometries with cross sections close to those estimated for the native solution phase structure were observed for the low charge states. For intermediate charge states, the compact geometries are metastable, and when collisionally heated they gradually unfold, through a series of well-defined intermediates. Only extended conformations are observed for the higher charge states, and they become more extended as the charge increases. The gas-phase conform...

Gas phase ion mobility measurements can resolve structural isomers for polyatomic ions and provide information about their geometries. A new experimental apparatus for performing high-resolution ion mobility measurements is described. The apparatus consists of a pulsed laser vaporization/desorption source coupled through an ion gate to a 63-cm-long drift tube. The ion gate is a critical component that prevents the diffusion of neutral species from the source into the drift tube. Ions travel along the drift tube under the influence of a uniform electric field. At the end of the drift tube some of the ions exit through a small aperture. They are focused into a quadrupole mass spectrometer, where they are mass analyzed, and then detected by an off-axis collision dynode and by dual microchannel plates. The apparatus is operated with a drift voltage of up to 14 000 V and a helium buffer gas pressure of around 500 Torr. The resolving power for ion mobility measurements is over an order of magnitude higher than has been achieved using conventional injected-ion drift tube techniques. Examples of the application of the new apparatus in resolving isomers of laser desorbed metallofullerenes, in studying silicon clusters generated by laser vaporization, and in following the isomerization of small nanocrystalline (NaCl)nCl− clusters as a function of temperature, are presented.

The ion-molecule reactions of thermalized iron-oxide cation FeO + with dihydrogen and methane have been studied by three different experimental techniques: Fourier transform ion cyclotron resonance (ICR), guided ion beam (GIB), and selected-ion flow tube (SIFT) mass spectrometry. Although these studies agree in a qualitative sense, i.e., FeO + brings about activation of H 2 and CH 4 with quite low efficiencies, there exists a considerable quantitative divergence as far as rate constants and branching ratios are concerned. The sources of error in these three related, but yet different experimental techniques are analyzed and critically reviewed. This error analysis brings the data to internal consistency with each other, once an accurate reference is used for calibration. In general, the rate constants obtained with the SIFT apparatus appear as the most accurate ones, while those obtained under ICR conditions are slightly too large, and the rate constants determined with the GIB instrument are somewhat lower than SIFT. However, the branching ratios for the formation of Fe + and FeOH + in the reaction of FeO + with methane are subject to more subtle effects. In the SIFT apparatus, termolecular stabilization of the intermediates causes differences from the ICR and GIB measurements, which were obtained under single-collision conditions.

In the structure of DL-proline, C5H9NO2, the molecules are connected via classical intermolecular N-H...O hydrogen bonds involving the amine and carboxyl groups [N...O = 2.7129 (15) and 2.8392 (16) A], and form chains along the b-axis direction and parallel to (-101). The chains are linked into sheets via weak non-classical hydrogen bonds. The conformation of the molecule and its packing are notably different from the monohydrated DL-proline form.

The combination of mass spectrometry and ion mobility spectrometry (IMS) employing a temperature-variable drift cell or a drift tube divided into sections to make IMS-IMS experiments possible allows information to be obtained about the molecular dynamics of polyatomic ions in the absence of a solvent. The experiments allow the investigation of structural changes of both activated and native ion populations on a timescale of 1–100 ms. Five different systems representing small and large, polar and nonpolar molecules, as well as noncovalent assemblies, are discussed in detail: a dinucleotide, a sodiated polyethylene glycol chain, the peptide bradykinin, the protein ubiquitin, and two types of peptide oligomers. Barriers to conformational interconversion can be obtained in favorable cases. In other cases, solution-like native structures can be observed, but care must be taken in the experimental protocols. The power of theoretical modeling is demonstrated.

Past experimental results and molecular dynamics simulations provide evidence that, under some conditions, electrospray ionization (ESI) of biomolecules produces ions that retain elements of solution phase structures. However, there is a dearth of information regarding the question raised by Breuker and McLafferty, "for how long, under what conditions, and to what extent, can solution structure be retained without solvent?" (Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 18145). Here, we use cryogenic ion mobility-mass spectrometry to experimentally probe the structural evolution of the undecapeptide substance P (SP) during the final stages of ESI. The results reveal that anhydrous SP conformers originate from evaporation of cluster ions, specifically, [SP + 2H](2+) (H2O)n (n = 0 to ∼50) and [SP + 3H](3+) (H2O)n (n = 0 to ∼30), and that major structural changes do not occur during the evaporative process. In the case of [SP + 3H](3+), the results demonstrate that a compact dehydrated conformer population can be kinetically trapped on the time scale of several milliseconds, even when an extended gas phase conformation is energetically favorable.

A novel overtone mobility spectrometry (OMS) instrument utilizing a gridless elimination mechanism and cooperative radio frequency confinement is described. The gridless elimination region uses a set of mobility-discriminating radial electric fields that are designed so that the frequency of field application results in selective transmission and elimination of ions. To neutralize ions with mobilities that do not match the field application frequency, active elimination regions radially defocus ions towards the lens walls. Concomitantly, a lens-dependent radio frequency waveform is applied to the transmission regions of the drift tube resulting in radial confinement for mobility-matched ions. Compared with prior techniques, which use many grids for ion elimination, the new gridless configuration substantially reduces indiscriminate ion losses. A description of the apparatus and elimination process, including detailed simulations showing how ions are transmitted and eliminated is presented. A prototype 28 cm long OMS instrument is shown to have a resolving power of 20 and is capable of attomole detection limits of a model peptide (angiotensin I) spiked into a complex mixture (in this case peptides generated from digestion of β-casein with trypsin).

Injected ion drift tube techniques, including ion mobility measurements and annealing and fragmentation studies, have been used to examine the isomers present for NbC,+ (n = 15-50) clusters. Isomers attributed to niobium-containing monocyclic and bicyclic rings, graphitic sheets, and metallofullerenes have been identified. Monocyclic rings, where the niobium atom appears to be either inserted into or bound to the outside of the ring, dominate for NbC,+ with n 22. Isomers assigned to bicyclic rings are first observed and become dominant around NbC22+. Unlike the bicyclic rings for C,' and Lac,+, the NbC,+ bicyclic rings do not anneal into monocyclic rings. They probably consist of two rings joined together by a niobium atom. An isomer attributed to NbC,+ graphitic sheets is present for n > 22 and becomes important for clusters with around 30 carbon atoms. Metallofullerenes are first observed for NbC28+ and become a major isomer for clusters with n 31. Both endohedral metallofullerenes and networked metallofullerenes (where the metal atom is part of the cage) have been identified. For clusters with more than around 30 carbon atoms the NbC,+ bicyclic rings can be annealed into metallofullerenes and, for the smaller ones, metal-containing graphitic sheets. The isomers observed for NbC,+ are similar to those found for pure C,+ and Lac,+, but the niobium atom has a substantial effect on the properties and the abundances of the different isomers.

Reaction kinetics of ground, Ti(a3F) and V(a4F), and excited, Ti(a5F) and V(a6D), states atoms with some simple molecules have been studied by a discharge-flow tube technique. Laser-induced fluorescence (LIF) was used to determine the concentration of the metal atoms as a function of the flow rate of the reactant molecule to obtain effective bimolecular rate constants. The rate constants of reactions with OX (X = O, N, N2) and H2S show strong inverse correlation with effective ionization potentials (I.P.-Eel, where I.P. and Eel are ionization potential and electronic energy of metal atom) of the metal atoms. This result suggests that the electron transfer mechanism plays an important role in these reactions. The large rate constants for electronic excited states can be explained by the crossing between ionic and flat neutral potential energy surfaces. The inefficient reaction rates measured for ground states can be explained by the repulsive nature of 4s2 configuration which can result in a potential barrier.

Oxidative modifications can have significant effects on protein structure in solution. Here, the structures and stabilities of oxidized ubiquitin ions electrosprayed from an aqueous solution (pH 2) are studied by ion mobility spectrometry-mass spectrometry (IMS-MS). IMS-MS has proven to be a valuable technique to assess gas phase and in many cases, solution structures. Herein, in vitro oxidation is performed by Fenton chemistry with Fe(II)/hydrogen peroxide. Most molecules in solution remain unmodified, whereas ∼20% of the population belongs to an M+16 Da oxidized species. Ions of low charge states (+7 and +8) show substantial variance in collision cross section distributions between unmodified and oxidized species. Novel and previously reported Gaussian conformers are used to model cross section distributions for +7 and +8 oxidized ubiquitin ions, respectively, in order to correlate variances in observed gas-phase distributions to changes in populations of solution states. Based on Gaussian modeling, oxidized ions of charge state +7 have an A-state conformation which is more populated for oxidized relative to unmodified ions. Oxidized ubiquitin ions of charge state +8 have a distribution of conformers arising from native-state ubiquitin and higher intensities of A- and U-state conformers relative to unmodified ions. This work provides evidence that incorporation of a single oxygen atom to ubiquitin leads to destabilization of the native state in an acidic solution (pH ∼2) and to unfolding of gas-phase compact structures.

Three disease phenotypes, Barrett’s esophagus (BE), high-grade dysplasia (HGD), esophageal adenocarcinoma (EAC), and a set of normal control (NC) serum samples are examined using a combination of ion mobility spectrometry (IMS), mass spectrometry (MS) and principal component analysis (PCA) techniques. Samples from a total of 136 individuals were examined, including: 7 characterized as BE, 12 as HGD, 56 as EAC and 61 as NC. In typical datasets it was possible to assign ~20 to 30 glycan ions based on MS measurements. Ion mobility distributions for these ions show multiple features. In some cases, such as the [S1H5N4+3Na]3+ and [S1F1H5N4+3Na]3+ glycan ions, the ratio of intensities of high-mobility features to low-mobility features vary significantly for different groups. The degree to which such variations in mobility profiles can be used to distinguish phenotypes is evaluated for eleven N-linked glycan ions. An outlier analysis on each sample class followed by an unsupervised PCA using a genetic algorithm for pattern recognition reveals that EAC samples are separated from NC samples based on 46 features originating from the 11-glycan composite IMS distribution.

Mathematical expressions for the analytical duty cycle associated with different overtones in overtone mobility spectrometry are derived from the widths of the transmitted packets of ions under different instrumental operating conditions. Support for these derivations is provided through ion trajectory simulations. The outcome of the theory and simulations indicates that under all operating conditions there exists a limit or maximum observable overtone that will result in ion transmission. Implications of these findings on experimental design are discussed.

A database of 1470 collision cross sections (666 doubly- and 804 triply-charged) of alkaline-earth-coordinated tryptic peptide ions [where the cation (M(2+)) correspond to Mg(2+), Ca(2+), or Ba(2+)] is presented. The utility of such an extensive set of measurements is illustrated by extraction of general properties of M(2+)-coordinated peptide structures. Specifically, we derive sets of intrinsic size parameters (ISPs) for individual amino acid residues for M(2+)-coordinated peptides. Comparison of these parameters with existing ISPs for protonated peptides suggests that M(2+) binding occurs primarily through interactions with specific polar aliphatic residues (Asp, Ser, and Thr) and the peptide backbone. A comparison of binding interactions for these alkaline-earth metals with interactions reported previously for alkali metals is provided. Finally, we describe a new analysis in which ISPs are used as probes for assessing peptide structure based on amino acid composition.

The isomers of the Man(7)GlcNAc(2) glycan obtained from bovine ribonuclease B have been characterized by ion mobility spectrometry-tandem mass spectrometry (IMS-MS/MS). In these experiments, [Man7 + 2Na](2+) precursors having different mobilities are selected by ion mobility spectrometry and analyzed by MS/MS techniques in an ion trap. The fragmentation spectra obtained for various precursor ions are specific, suggesting the isolation or enrichment of different glycan isomers. One fragment ion with a mass-to-charge ratio (m/z) of 903.8 is found to correspond to the loss of an internal mannose residue of a specific isomer. Extracted fragment ion drift time distributions (XFIDTDs) yield distinctive precursor ion drift time profiles indicating the existence of four separate isomers as proposed previously.

Ion mobility/mass spectrometry techniques are used to study the chiral preferences of small proline clusters (containing 2 to 23 proline monomers) produced by electrospray ionization. By varying the composition of the electrospray solution from enantiomerically pure (100% L or 100% D) to racemic (50:50 L:D), it is possible to delineate which cluster sizes prefer homochiral (resolved) or heterochiral (antiresolved) compositions. The results show a remarkable oscillation in chiral preference. Singly protonated clusters, [xPro+H](+) (where x corresponds to the number of prolines), favor homochiral assemblies (for x = 4, 6, 11 and 12); heterochiral structures are preferred (although the preferences are not as strong) for x = 5 and 7. Larger, doubly protonated clusters [xPro+2H](2+) favor homochiral assemblies for x = 18, 19, and 23 and heterochiral structures for x = 14, 16, 17, 20, 21, and 22. Some of the variations that are observed can be rationalized through simple structures that would lead to especially stable geometries. It is suggested that some antiresolved clusters, such as [22Pro+2H](2+), may be comprised of resolved D- and L-proline domains.

Ion mobility/mass spectrometry techniques are used to investigate the dissociation of the small proline cluster [14Pro+2H](2+) produced by electrospray ionization. While this cluster is known to prefer heterochiral compositions (i.e., mixed L- and D-compositions, J. Phys. Chem. A, submitted for publication), it is possible to produce homochiral forms by electrospraying solutions containing only L- or D-proline. Differences in the measured cross sections for [14Pro+2H](2+) produced from enantiomerically pure (100% l or 100% d) or racemic (50:50 l/d) solutions indicate that homochiral and heterochiral clusters have different structures. Upon low-energy collisional activation, both the heterochiral and homochiral doubly charged structures evaporate neutral proline monomers, resulting in the formation of [xPro+2H](2+) ions (where x = 9-13). At higher activation energies, there is evidence that these smaller clusters (primarily [10Pro+2H](2+)) fission to produce [xPro+H](+) (where x = 1-6). Analysis of product ion intensities reveals a strong chiral preference associated with fissioning. Products of evaporation also show a chiral dependence but to a lesser extent.

A new method is presented for constructing ion mobility distributions of precursor ions based upon the extraction of drift time distributions that are monitored for selected fragment ions. The approach is demonstrated with a recently designed instrument that combines ion mobility spectrometry (IMS) with ion trap mass spectrometry (MS) and ion fragmentation, as shown in a recent publication [J. Am. Soc. Mass Spectrom. 22 (2011) 1477–1485]. Here, we illustrate the method by examining selected charge states of electrosprayed ubiquitin ions, an extract from diesel fuel, and a mixture of phosphorylated peptide isomers. For ubiquitin ions, extraction of all drift times over small mass-to-charge (m/z) ranges corresponding to unique fragments of a given charge state allows the determination of precursor ion mobility distributions. A second example of the utility of the approach includes the distinguishing of precursor ion mobility distributions for isobaric, basic components from commercially available diesel fuel. Extraction of data for a single fragment ion is sufficient to distinguish the precursor ion mobility distribution of cycloalkyl-pyridine derivatives from pyrindan derivatives. Finally, the method is applied for the analysis of phosphopeptide isomers (LFpTGHPESLER and LFTGHPEpSLER) in a mixture. The approach alleviates several analytical challenges that include separation and characterization of species having similar (or identical) m/z values within complex mixtures.

The reactions of Sc[sup +] and Ti[sup +] with D[sub 2]O are studied as a function of translational energy in a guided ion beam tandem mass spectrometer. Both ions react to form three ionic products. MD[sup +], MO[sup +], and MOD[sup +]. MO[sup +] formation occurs exothermically by dehydrogenation of D[sub 2]O, even though this reaction is spin forbidden for reaction of ground state M[sup 1] in both systems. In the titanium system, the effect of electronic energy on product formation is probed by varying the conditions used for forming Ti[sup +]. The a[sup 2]F state of Ti[sup +] reacts much more efficiently than the a[sup 4]F ground state at forming all three product ions. The relative reaction efficiencies are consistent with a recent study of the reverse reactions of MO[sup +] + D[sub 2]. State-specific cross sections and the competition between the various products indicate that the reactions occur primarily through a low-spin state of a D-M[sup +]-OD intermediate. Thresholds of the cross sections for endothermic formation of all three product ions are consistent with values calculated by using previously measured bond energies. 44 refs., 5 figs., 3 tabs.

The mechanisms and energetics involved in the conversion of CH{sub 4} to CH{sub 3}OH by CoO{sup +} are examined by using guided ion beam mass spectrometry. The forward and reverse reactions, CO{sup +} + CH{sub 4} CO{sup +} + Ch{sub 3}OH, the collisional activation of Co{sup +}(CH{sub 3}OH), and the related reactions, CoO{sup +} + D{sub 2} Co{sup +} + D{sub 2}O, are studied. It is found that the oxidations of methane and D{sub 2} by CoO{sup +}, both exothermic reactions, do not occur until overcoming activation barriers of 0.56 x 0.08 and 0.75 x 0.04 eV, respectively. The behavior of the forward and reverse reactions in both systems is consistent with reactions that proceed via the insertion intermediates R-Co{sup +}-OH, where R = CH{sub 3} or H. The barrier is probably attributable to a four-centered transition state associated with addition of RH across the CoO{sup +} bond. In the Co{sup +} + CH{sub 3}OH system (where CH{sub 3}OD labeled reactant is used), reactions explained by initial C-H and O-H activation are also observed. The reaction mechanisms and potential energy surfaces for these systems are derived and discussed. Phase space theory calculations are used to help verify these details formore » the CoO{sup +} + D{sub 2} system. Thermochemistry for several species including CoOH{sup +}, CoD{sup +}, CoH, CoCH{sub 3}{sup +}, Co{sup +}(CH{sub 3}OD), CoOCH{sub 3}{sup +}, and possibly OCoCH{sub 3}{sup +} is derived from measurements of reaction thresholds. 59 refs., 8 figs., 4 tabs.« less

Reactions of Fe+ with DzO and FeO+ with Dz are studied as a function of translational energy in a guided-ion beam tandem mass spectrometer. In the former system, the only products observed from single-collision events are FeD+ and FeOD+. These products are formed in endothermic reactions. At low energies, the FeODZ+ adduct is also observed, a result of secondary stabilizing collisions with D2O. Results for Fe+ produced in two different sources are analyzed to yield state-specific cross sections for reaction of the a6D ground and a4F first excited states of Fe+. In the reaction of FeO+ with D2, three ionic products (Fe+, FeD+, and FeOD+) are observed. An inefficient exothermic process that forms Fe+ + DzO is observed, consistent with known thermochemistry; however, formation of Fe+ + DzO also occurs via another more efficient pathway that involves a reaction barrier of -0.6 eV. The FeOD+ channel also proceeds via a pathway involving a reaction barrier of the same energy. Results for both the Fe+ + DzO and FeO+ + Dz systems are used to derive potential energy surfaces for these systems.

Altered branching and aberrant expression of N-linked glycans is known to be associated with disease states such as cancer. However, the complexity of determining such variations hinders the development of specific glycomic approaches for assessing disease states. Here, we examine a combination of ion mobility spectrometry (IMS) and mass spectrometry (MS) measurements, with principal component analysis (PCA) for characterizing serum N-linked glycans from 81 individuals: 28 with cirrhosis of the liver, 25 with liver cancer, and 28 apparently healthy. Supervised PCA of combined ion-mobility profiles for several, to as many as 10 different mass-to-charge ratios for glycan ions, improves the delineation of diseased states. This extends an earlier study [J. Proteome Res.2008, 7, 1109-1117] of isomers associated with a single glycan (S(1)H(5)N(4)) in which PCA analysis of the IMS profiles appeared to differentiate the liver cancer group from the other samples. Although performed on a limited number of test subjects, the combination of IMS-MS for different combinations of ions and multivariate PCA analysis shows promise for characterizing disease states.

Ion mobility and mass spectrometry measurements have been used to examine the populations of different solution structures of the nonapeptide bradykinin. Over the range of solution compositions studied, from 0:100 to 100:0 methanol:water and 0:100 to 90:10 dioxane:water, evidence for ten independent populations of bradykinin structures in solution is found. In some solutions as many as eight structures may coexist. The solution populations are substantially different than the gas-phase equilibrium distribution of ions, which exhibits only three distinct states. Such a large number of coexisting structures explains the inability of traditional methods of characterization such as nuclear magnetic resonance spectroscopy and crystallography to determine detailed structural features for some regions of this peptide.

A new instrument that combines ion mobility spectrometry (IMS) separations with tandem mass spectrometry (MS(n)) is described. Ion fragmentation is achieved with vacuum ultraviolet photodissociation (VUV PD) and/or collision-induced dissociation (CID). The instrument is comprised of an approximately 1 m long drift tube connected to a linear trap that has been interfaced to a pulsed F(2) laser (157 nm). Ion gates positioned in the front and the back of the primary drift region allow for mobility selection of specific ions prior to their storage in the ion trap, mass analysis, and fragmentation. The ion characterization advantages of the new instrument are demonstrated with the analysis of the isomeric trisaccharides, melezitose and raffinose. Mobility separation of precursor ions provides a means of separating the isomers and subsequent VUV PD generates unique fragments allowing them to be distinguished.

The binding properties of a peptidoglycan recognition protein are translated via combinatorial chemistry into short peptides. Non-adjacent histidine, tyrosine, and arginine residues in the protein’s binding cleft that associate specifically with the glycan moiety of a peptidoglycan substrate are incorporated into linear sequences creating a library of 27 candidate tripeptide reagents (three possible residues permutated across three positions). Upon electrospraying the peptide library and carbohydrate mixtures, some noncovalent complexes are observed. The binding efficiencies of the peptides vary according to their amino acid composition as well as the disaccharide linkage and carbohydrate ring-type. In addition to providing a charge-carrier for the carbohydrate, peptide reagents can also be used to differentiate carbohydrate isomers by ion mobility spectrometry. The utility of these peptide reagents as a means of enhancing ion mobility analysis of carbohydrates is illustrated by examining four glucose-containing disaccharide isomers, including a pair that is not resolved by ion mobility alone. The specificity and stoichiometry of the peptide–carbohydrate complexes are also investigated. Trihistidine demonstrates both suitable binding efficiency and successful resolution of disaccharides isomers, suggesting it may be a useful reagent in IMS analyses of carbohydrates.

Complexation of a series of related amino compounds by 18-crown-6 ether (18C6) is studied as a means of improving the resolution of mixtures by combinations of ion mobility spectrometry (IMS) and mass spectrometry (MS) techniques. Mixtures of the isomeric amines n-octylamine (NOA), dibutylamine (DBA), and diisopropylethylamine (DIPEA) were electrosprayed to produce gaseous [M + H](+) ions. These species have overlapping mobilities and are not resolved by IMS. Addition of 18C6 yields [M + 18C6 + H](+) ion complexes that are resolved by IMS. In subsequent experiments, [M + 18C6 + H](+) ion complexes are separated according to their mobilities and specific species are selected and exposed to collisional activation. This analysis yields dissociation voltages that are inversely correlated with the number of separate substitutions on the nitrogen atom of the amino compounds; dissociation voltages of ~40, ~90, and ~150 V are obtained for the tri-, di-, and mono-substituted amino compounds DIPEA, DBA, and NOA, respectively. For these complexes, an inverse correlation is also observed with respect to the gas-phase basicities (GB) of the amino compounds (964, 935, and 895 kJ mol(-1), respectively). Studies of 18C6 complexes with a series of n-alkylamines (C( n )H(2n+3)N where n=3 to 18, respectively) show that dissociation voltages increase systematically (from ~140 to ~190 V) under the conditions employed. The sensitivity to collision energy provides an additional means of distinguishing between classes of compounds. The approach is extended as a means of separating nitrogen-containing compounds from petroleum.

The origin of non-integer overtone peaks in overtone mobility spectrometry (OMS) spectra is investigated by ion trajectory simulations. Simulations indicate that these OMS features arise from higher-order overtone series. An empirically-derived formula is presented as a means of describing the positions of peaks. The new equation makes it possible to determine collision cross sections from any OMS peak. Additionally, it is extended as a means of predicting the resolving power for any peak in an OMS distribution.

Modification of ubiquitin, a key cellular regulatory polypeptide of 76 amino acids, to polyubiquitin conjugates by lysine-specific isopeptide linkage at one of its seven lysine residues has been recognized as a central pathway determining its biochemical properties and cellular functions. Structural details and differences of distinct lysine-isopeptidyl ubiquitin conjugates that reflect their different functions and reactivities, however, are only partially understood. Ion mobility spectrometry (IMS) combined with mass spectrometry (MS) has recently emerged as a powerful tool for probing conformations and topology involved in protein interactions by an electric field-driven separation of polypeptide ions through a drift gas. Here we report the conformational characterization and differentiation of Lys63- and Lys48-linked ubiquitin conjugates by IMS–MS. Lys63- and Lys48-linked di-ubiquitin conjugates were prepared by recombinant bacterial expression and by chemical synthesis using a specific chemical ligation strategy, and characterized by high-resolution Fourier transform ion cyclotron resonance mass spectrometry, circular dichroism spectroscopy, and molecular modeling. IMS–MS was found to be an effective tool for the identification of structural differences of ubiquitin complexes in the gas phase. The comparison of collision cross-sections of Lys63- and Lys48-linked di-ubiquitin conjugates showed a more elongated conformation of Lys63-linked di-ubiquitin. In contrast, the Lys48-linked di-ubiquitin conjugate showed a more compact conformation. The IMS-MS results are consistent with published structural data and a comparative molecular modeling study of the Lys63- and Lys48-linked conjugates. The results presented here suggest IMS techniques can provide information that complements MS measurements in differentiating higher-order polyubiquitins and other isomeric protein linkages.

The sequential bond energies of Cr(CO) x + , x=1-6, are determined by collision-induced dissociation in a guided ion beam tandem mass spectrometer. Values for the 0 K bond energies (in eV) are determined to be D(Cr + -CO)=0.93±0.04, D[(CO)Cr + -CO]=0.98±0.03, D[(CO) 2 Cr + -CO]=0.56±0.06, D[(CO) 3 Cr + -CO]=0.53±0.08, D[(CO) 4 Cr + -CO]=0.64±0.03, and D[(CO) 5 Cr + -CO]=1.35±0.08

The homolytic bond dissociation energies of the transition‐metal neutral hydrides, Do(M–H) (M=V, Cr), are experimentally determined by using guided ion beam tandem mass spectrometry to measure the kinetic energy dependence of the endothermic hydride abstraction reactions of M+ with mono‐, di‐, and trimethylamine. From the thresholds of these reactions, we derive the 0 K values of Do(V–H)=2.13±0.07 eV and Do(Cr–H)=1.93±0.07 eV. This thermochemistry is compared with theoretical values and previous experimental results.

A new operational mode for an ion cyclotron mobility spectrometry instrument is explored as a possible means of performing high-resolution separations. The approach is based on oscillating fields that are applied to segmented regions of a circular drift tube. Ions with mobilities that are resonant with the frequency of field application are transmitted while nonresonant species are eliminated. An ion mobility spectrum is obtained by scanning the drift field application frequency. The approach is demonstrated by examining mixtures of ions produced by electrospraying the substance P peptide, as well as a mixture of tryptic peptides obtained by enzymatic digestion of cytochrome c. Drift field application frequency scans of substance P peptide ions show that it is possible to separate [M+2H](2+) ions, and compact and elongated forms of [M+3H](3+) ions. The resolution of different ions is related to the number of cycles for the analysis. At high cycle numbers (>50 3/4 or a drift length of 9242.03 cm) values of the resolving power can exceed 300 with a maximum resolving power of ∼400. The ability to tune the resolving power of a mobility-based separation by varying the ion cycle number has substantial analytical utility.

The reactions of Sc+, Ti+, and V+ with CO are studied as a function of translational energy in a guided‐ion‐beam tandem mass spectrometer. Formation of both metal‐carbide and metal‐oxide ions are observed and rationalized by a direct atom abstraction mechanism. At high energies, the ScC+ and ScO+ cross sections exhibit additional features that are unusual but can be explained by an impulsive pairwise mechanism and formation of excited‐state product ions, respectively. Thresholds of the reaction cross sections are interpreted to give the 0 K bond energies (in eV) D0(ScC+)=3.34±0.06, D0(TiC+)=4.05±0.24, D0(VC+)=3.87±0.14, D0(ScO+)=7.11±0.08, D0(TiO+)=6.88±0.07, and D0(VO+)=5.81±0.17. Additional studies are used to help verify the bond energy for ScO+ and yield a recommended value of 7.14±0.11 eV. The nature of the bonding in MO+ and MC+ is discussed and compared for these three metal ions.

Cross sections for the reactions of ScO[sup +], TiO[sup +], and VO[sup +] with D[sub 2] have been measured as a function of translational energy in a guided ion beam tandem mass spectrometer. All three MO[sup +] ions react with D[sub 2] to form three products, M[sup +], MD[sup +], and MOD[sup +], in endothermic processes. Production of M[sup +] in all systems is accompanied by formation of water. The energetics measured for these reactions correspond to production of M[sup +] primarily in an excited low-spin electronic state. Formation of ground-state M[sup +] is also observed, even though this reaction channel does not conserve spin. Reaction mechanisms and qualitative potential energy surfaces are proposed to explain this behavior. Thresholds for the formation of MOD[sup +] in these systems are interpreted to give the 0 K bond energies, D[degree](Sc[sup +]-OD) = 5.17 [+-] 0.09, D[degree](Ti[sup +]-OD) = 4.82 [+-] 0.12, and D[degree](V[sup +]-OD) = 4.50 [+-] 0.15, all in electronvolts. The large bond strengths for the M[sup +]-OD species indicate that the lone pairs of electrons on the oxygen atom are involved in the bonding. 48 refs., 9 figs., 4 tabs.

A summary of recent studies of the geometries and structural dynamics of pure carbon and metal containing carbon clusters is given. For pure carbon clusters the bicyclic and polycyclic rings can be converted into either monocyclic rings or fullerenes. Formation of the monocyclic rings is driven by a decrease in the strain energy, while fullerene formation is driven by formation of the stable fullerene cage. Mechanisms which explain the formation of bicyclic and polycyclic rings, as well as the conversion of these into monocyclic rings and fullerenes are discussed.© (1994) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Although nonnative protein conformations, including intermediates along the folding pathway and kinetically trapped misfolded species that disfavor the native state, are rarely isolated in the solution phase, they are often stable in the gas phase, where macromolecular ions from electrospray ionization can exist in varying charge states. Differences in the structures of nonnative conformations in the gas phase are often large enough to allow different shapes and charge states to be separated because of differences in their mobilities through a gas. Moreover, gentle collisional activation can be used to induce structural transformations. These new structures often have different mobilities. Thus, there is the possibility of developing a multidimensional separation that takes advantage of structural differences of multiple stable states. This review discusses how nonnative states differ in the gas phase compared with solution and presents an overview of early attempts to utilize and manipulate structures in order to develop ion mobility spectrometry as a rapid and sensitive technique for separating complex mixtures of biomolecules prior to mass spectrometry.

The recent development of electrospray ionization (ESI)1 and matrix-assisted laser desorption ionization (MALDI)2 sources for creation of macromolecular ions has brought about a renaissance in the field of mass spectrometry (MS). It is now common to determine molecular weights of large molecules such as proteins to within a few daltons (or less)! MS-based methods for sequencing are proliferating and replacing (or complementing) traditional biochemical methods for many applications. However, analysis of covalent structure is only part of the story. A number of studies have investigated the use of MS strategies for examining noncovalent biomolecular structure. Mounting evidence suggests that some conformational properties of biomolecules in solution are preserved during the ionization process and persist over the transient time that ions exist in mass spectrometers. The persistence of solution-like structure in the gas phase provides opportunities to examine conformation and folding as well as binding interactions (e.g., enzyme-substrate, receptor-ligand interactions) in an entirely new environment. In the past 5 years, a new field involving structural studies of biomolecular ions has emerged. At first glance, the relevance of biomolecular ion structure in the gas phase to studies of functional molecules in solution may seem far removed, perhaps not unlike questions about the relevance of structural measurements of protein crystals 20-30 years ago. Studies of biomolecular ions in the gas phase provide information about the nature of conformation in the absence of solvent; thus, the roles of intramolecular interactions and solvent can be discerned by comparison of similarities between gasand solutionphase structure. Additionally, the utility of MS strategies for sampling noncovalent binding between molecules in solution hinge on a fragile balance of interactions associated with the assembled pair as the ions travel through the mass analyzer. As these strategies become widely used, the differentiation 3037 Chem. Rev. 1999, 99, 3037−3079