Proteome Mapping of the Human Pancreatic Islet Microenvironment Reveals Endocrine-Exocrine Signaling Sphere of Influence.

The need for a clinically accessible method with the ability to match protein activity within heterogeneous tissues is currently unmet by existing technologies. Our proteomics sample preparation platform, named microPOTS (Microdroplet Processing in One pot for Trace Samples), can be used to measure relative protein abundance in micron-scale samples alongside the spatial location of each measurement, thereby tying biologically interesting proteins and pathways to distinct regions. However, given the smaller pixel/voxel number and amount of tissue measured, standard mass spectrometric analysis pipelines have proven inadequate. Here we describe how existing computational approaches can be adapted to focus on the specific biological questions asked in spatial proteomics experiments. We apply this approach to present an unbiased characterization of the human islet microenvironment comprising the entire complex array of cell types involved while maintaining spatial information and the degree of the islet's sphere of influence. We identify specific functional activity unique to the pancreatic islet cells and demonstrate how far their signature can be detected in the adjacent tissue. Our results show that we can distinguish pancreatic islet cells from the neighboring exocrine tissue environment, recapitulate known biological functions of islet cells, and identify a spatial gradient in the expression of RNA processing proteins within the islet microenvironment.

Water-Assisted Proton Transfer in the Sequential Hydration of Benzonitrile Radical Cation C6H5CN•+(H2O)n: Transition to Hydrated Distonic Cation •C6H4CNH+(H2O)n with n ≥ 4.

The stepwise hydration of the benzonitrile•+ radical cation with one-seven H2O molecules was investigated experimentally and computationally with density functional theory in C6H5CN•+(H2O)n clusters. The stepwise binding energies (ΔHn-1,n°) were determined by equilibrium measurements for C6H5CN•+(H2O) and for •C6H4CNH+(H2O)n with n = 5, 6, and 7 to be 8.8 and 11.3, 11.0, and 10.0 kcal/mol, respectively. The populations of n = 2 and 3 of the C6H5CN•+(H2O)n clusters were observed only in trace abundance due to fast depletion processes leading to the formation of the hydrated distonic cations •C6H4CNH+(H2O)n with n = 4-7. The observed transition occurs between conventional radical cations hydrated on the ring in C6H5CN•+(H2O)n clusters with n = 1-3 and the protonated radical •C6H4CNH+ (distonic ion) formed by a proton transfer to the CN nitrogen and ionic hydrogen bonding to water molecules in •C6H4CNH+(H2O)n clusters with n = 4-7. The measured binding energy of the hydrated ion C6H5CN•+(H2O) (8.8 kcal/mol) is similar to that of the hydrated benzene radical cation (8.5 kcal/mol) that involves a relatively weak CHδ+···O hydrogen bonding interaction. Also, the measured binding energies of the •C6H4CNH+(H2O)n clusters with n = 5-7 are similar to those of the protonated benzonitrile (methanol)n clusters [C6H5CNH+(CH3OH)n, n = 5-7] that involve CNH+···O ionic hydrogen bonds. The proton shift from the para-•C ring carbon to the nitrogen of the benzonitrile radical cation is endothermic without solvent but thermoneutral for n = 1 and exothermic for n = 2-4 in C6H5CN•+(H2O)n clusters to form the distonic •C6H4CN···H+(OH2)n clusters. The distonic clusters •C6H4CN···H+(OH2)n constitute a new class of structures in radical ion/solvent clusters.

Activity-Based Protein Profiling of Chitin Catabolism.

The microbial catabolism of chitin, an abundant and ubiquitous environmental organic polymer, is a fundamental cog in terrestrial and aquatic carbon and nitrogen cycles. Despite the importance of this critical bio-geochemical function, there is a limited understanding of the synergy between the various hydrolytic and accessory enzymes involved in chitin catabolism. To address this deficit, we synthesized activity-based probes (ABPs) designed to target active chitinolytic enzymes by modifying the chitin subunits N-acetyl glucosamine and chitotriose. The ABPs were used to determine the active complement of chitinolytic enzymes produced over time by the soil bacterium Cellvibrio japonicus treated with various C substrates. We demonstrate the utility of these ABPs in determining the synergy between various enzymes involved in chitin catabolism. The strategy can be used to gain molecular-level insights that can be used to better understand microbial roles in soil bio-geochemical cycling in the face of a changing climate.

Assessing Collision Cross Section Calibration Strategies for Traveling Wave-Based Ion Mobility Separations in Structures for Lossless Ion Manipulations.

The collision cross section (CCS) is an important property that aids in the structural characterization of molecules. Here, we investigated the CCS calibration accuracy with traveling wave ion mobility spectrometry (TWIMS) separations in structures for lossless ion manipulations (SLIM) using three sets of calibrants. A series of singly negatively charged phospholipids and bile acids were calibrated in nitrogen buffer gas using two different TW waveform profiles (square and sine) and amplitudes (20, 25, and 30 V0-p). The calibration errors for the three calibrant sets (Agilent tuning mixture, polyalanine, and one assembled in-house) showed negligible differences using a sine-shaped TW waveform. Calibration errors were all within 1-2% of the drift tube ion mobility spectrometry (DTIMS) measurements, with lower errors for sine waveforms, presumably due to the lower average and maximum fields experienced by ions. Finally, ultrahigh-resolution multipass (long path length) SLIM TWIMS separations demonstrated improved CCS calibration for phospholipid and bile acid isomers.

Rapid and Simultaneous Characterization of Drug Conjugation in Heavy and Light Chains of a Monoclonal Antibody Revealed by High-Resolution Ion Mobility Separations in SLIM.

Antibody-drug conjugates (ADCs) have recently gained traction in the biomedical community due to their promise for human therapeutics and an alternative to chemotherapy for cancer. Crucial metrics for ADC efficacy, safety, and selectivity are their drug-antibody ratios (DARs). However, DAR characterization (i.e., determining the average number of conjugated drugs on the antibody) through analytical methods remains challenging due to the heterogeneity of drug conjugation as well as the numerous post-translational modifications possible in the monoclonal antibody. Herein, we report on the use of high-resolution ion mobility spectrometry separations in structures for lossless ion manipulations coupled to mass spectrometry (SLIM IMS-MS) for the rapid and simultaneous characterization of the drug load profile (i.e., stoichiometric distribution of the number of conjugated drugs present on the mAb), determination of the weighted average DAR in both the heavy and light chains of a model antibody-drug conjugate, and calculation of the overall DAR of the ADC. After chemical reduction of the ADC and a subsequent 31.5 m SLIM IMS separation, the various drug-bound antibody species could be well resolved for both chains. We also show significantly higher resolution separations were possible for these large ions with SLIM IMS as compared to ones performed on a commercially available (1 m) drift tube IMS-MS platform. We expect high-resolution SLIM IMS separations will augment the existing toolbox for ADC characterization, particularly to enable the rapid optimization of DAR for a given ADC and thus better understand its potential toxicity and potency.

Ion Mobility Spectrometry with High Ion Utilization Efficiency Using Traveling Wave-Based Structures for Lossless Ion Manipulations.

Ion packets introduced from gates, ion funnel traps, and other conventional ion injection mechanisms produce ion pulse widths typically around a few microseconds or less for ion mobility spectrometry (IMS)-based separations on the order of 100 milliseconds. When such ion injection techniques are coupled with ultralong path length traveling wave (TW)-based IMS separations (i.e., on the order of seconds) using structures for lossless ion manipulations (SLIMs), typically very low ion utilization efficiency is achieved for continuous ion sources [e.g., electrospray ionization (ESI)]. Even with the ability to trap and accumulate much larger populations of ions than being conventionally feasible over longer time periods in SLIM devices, the subsequent long separations lead to overall low ion utilization. Here, we report the use of a highly flexible SLIM arrangement, enabling concurrent ion accumulation and separation and achieving near-complete ion utilization with ESI. We characterize the ion accumulation process in SLIM, demonstrate >98% ion utilization, and show both increased signal intensities and measurement throughput. This approach is envisioned to have broad utility to applications, for example, involving the fast detection of trace chemical species.

Traveling-Wave-Based Electrodynamic Switch for Concurrent Dual-Polarity Ion Manipulations in Structures for Lossless Ion Manipulations.

We describe the development of a dual-polarity traveling-wave (TW) structures for lossless ion manipulations (SLIM) ion mobility spectrometry (IMS) device capable of switching both positive and negative ions that are traveling simultaneously along the same path to different regions of the SLIM. Through simulations, the routing efficiency of the SLIM TW switch was compared to a SLIM direct-current-based (DC) switch developed previously for IMS-MS. We also report on the initial experimental evaluation of a dual-polarity SLIM platform, which uses the TW-based ion switch to achieve higher resolution multipass serpentine ultralong path with extended routing (SUPER) IMS separations. Overall, these results show that the dual-polarity TW switch is not only as effective as DC switching in terms of routing efficiency but also is agnostic to the polarity of the ions being routed.

Separation of ß-Amyloid Tryptic Peptide Species with Isomerized and Racemized l-Aspartic Residues with Ion Mobility in Structures for Lossless Ion Manipulations.

Accumulation of ß-amyloid (Aß) is one of the hallmarks of Alzheimer's disease. The deposition of ß-amyloid plaques is likely to start years in advance of manifestation of clinical symptoms, although the exact timing is unknown. Over the years, Aß peptides undergo both post-translational modification and stereoisomerization. Analysis of the resulting stereoisomers is particularly challenging because of their identical elemental composition and similar physicochemical properties. Herein, we have utilized our recently developed structures for lossless ion manipulations ion mobility-mass spectrometry platform (SLIM IM-MS), in conjunction with serpentine ultralong path with extended routing (SUPER), to baseline resolve four distinct sets of Aß17-28 tryptic peptide epimers on a rapid (∼1 s) time scale. We discovered that sodium adduct ions, [M + H + Na]2+, allowed baseline SLIM SUPER IM resolution for all Aß epimer sets assessed, while such baseline separations were unachievable for their [M + 2H]2+ doubly protonated ions.

SLIM Ultrahigh Resolution Ion Mobility Spectrometry Separations of Isotopologues and Isotopomers Reveal Mobility Shifts due to Mass Distribution Changes.

We report on separations of ion isotopologues and isotopomers using ultrahigh-resolution traveling wave-based Structures for Lossless Ion Manipulations with serpentine ultralong path and extended routing ion mobility spectrometry coupled to mass spectrometry (SLIM SUPER IMS-MS). Mobility separations of ions from the naturally occurring ion isotopic envelopes (e.g., [M], [M+1], [M+2], ... ions) showed the first and second isotopic peaks (i.e., [M+1] and [M+2]) for various tetraalkylammonium ions could be resolved from their respective monoisotopic ion peak ([M]) after SLIM SUPER IMS with resolving powers of ∼400-600. Similar separations were obtained for other compounds (e.g., tetrapeptide ions). Greater separation was obtained using argon versus helium drift gas, as expected from the greater reduced mass contribution to ion mobility described by the Mason-Schamp relationship. To more directly explore the role of isotopic substitutions, we studied a mixture of specific isotopically substituted (15N, 13C, and 2H) protonated arginine isotopologues. While the separations in nitrogen were primarily due to their reduced mass differences, similar to the naturally occurring isotopologues, their separations in helium, where higher resolving powers could also be achieved, revealed distinct additional relative mobility shifts. These shifts appeared correlated, after correction for the reduced mass contribution, with changes in the ion center of mass due to the different locations of heavy atom substitutions. The origin of these apparent mass distribution-induced mobility shifts was then further explored using a mixture of Iodoacetyl Tandem Mass Tag (iodoTMT) isotopomers (i.e., each having the same exact mass, but with different isotopic substitution sites). Again, the observed mobility shifts appeared correlated with changes in the ion center of mass leading to multiple monoisotopic mobilities being observed for some isotopomers (up to a ∼0.04% difference in mobility). These mobility shifts thus appear to reflect details of the ion structure, derived from the changes due to ion rotation impacting collision frequency or momentum transfer, and highlight the potential for new approaches for ion structural characterization.

Isolation of Tryptanthrin and Reassessment of Evidence for Its Isobaric Isostere Wrightiadione in Plants of the Wrightia Genus.

A series of Wrightia hanleyi extracts was screened for activity against Mycobacterium tuberculosis H37Rv. One active fraction contained a compound that initially appeared to be either the isoflavonoid wrightiadione or the alkaloid tryptanthrin, both of which have been previously reported in other Wrightia species. Characterization by NMR and MS, as well as evaluation of the literature describing these compounds, led to the conclusion that wrightiadione (1) was misidentified in the first report of its isolation from W. tomentosa in 1992 and again in 2015 when reported in W. pubescens and W. religiosa. Instead, the molecule described in these reports and in the present work is almost certainly the isobaric (same nominal mass) and isosteric (same number of atoms, valency, and shape) tryptanthrin (2), a well-known quinazolinone alkaloid found in a variety of plants including Wrightia species. Tryptanthrin (2) is also accessible synthetically via several routes and has been thoroughly characterized. Wrightiadione (1) has been synthesized and characterized and may have useful biological activity; however, this compound can no longer be said to be known to exist in Nature. To our knowledge, this misidentification of wrightiadione (1) has heretofore been unrecognized.

Distinguishing enantiomeric amino acids with chiral cyclodextrin adducts and structures for lossless ion manipulations.

Enantiomeric molecular evaluations remain an enormous challenge for current analytical techniques. To date, derivatization strategies and long separation times are generally required in these studies, and the development and implementation of new approaches are needed to increase speed and distinguish currently unresolvable compounds. Herein, we describe a method using chiral cyclodextrin adducts and structures for lossless ion manipulations (SLIM) and serpentine ultralong path with extended routing (SUPER) ion mobility (IM) to achieve rapid, high resolution separations of d and l enantiomeric amino acids. In the analyses, a chiral cyclodextrin is added to each sample. Two cyclodextrins were found to complex each amino acid molecule (i.e. potentially sandwiching the amino acid in their cavities) and forming host-guest noncovalent complexes that were distinct for each d and l amino acid pair studied and thus separable with IM in SLIM devices. The SLIM was also used to accumulate much larger ion populations than previously feasible for evaluation and therefore allow enantiomeric measurements of higher sensitivity, with gains in resolution from our ultralong path separation capabilities, than previously reported by any other IM-based approach.

Nucleophilic Aromatic Addition in Ionizing Environments: Observation and Analysis of New C-N Valence Bonds in Complexes between Naphthalene Radical Cation and Pyridine.

Radical organic ions can be stabilized by complexation with neutral organics via interactions that can resemble chemical bonds, but with much diminished bond energies. Those interactions are a key factor in cluster growth and polymerization reactions in ionizing environments such as regions of the interstellar medium and solar nebulae. Such radical cation complexes between naphthalene (Naph) and pyridine (Pyr) are characterized using mass-selected ion mobility experiments. The measured enthalpy of binding of the Naph+•(Pyr) heterodimer (20.9 kcal/mol) exceeds that of the Naph+•(Naph) homodimer (17.8 kcal/mol). The addition of 1-3 more pyridine molecules to the Naph+•(Pyr) heterodimer gives 10-11 kcal/mol increments in binding enthalpy. A rich array of Naph+•(Pyr) isomers are characterized by electronic structure calculations. The calculated Boltzmann distribution at 400 K yields an enthalpy of binding in reasonable agreement with experiment. The global minimum is a distonic cation formed by Pyr attack on Naph+• at the α-carbon, changing its hybridization from sp2 to distorted sp3. The measured collision cross section in helium for the Naph+•(Pyr) heterodimer of 84.9 ± 2.5 Å2 at 302 K agrees well with calculated angle-averaged cross sections (83.9-85.1 Å2 at 302 K) of the lowest energy distonic structures. A remarkable 16 kcal/mol increase in the binding energy between Naph+•(Pyr) and Bz+•(Pyr) (Bz is benzene) is understood by energy decomposition analysis. A similar increase in binding from Naph+•(NH3) to Naph+•(Pyr) (as well as between Bz+•(NH3) and Bz+•(Pyr)) is likewise rationalized.

Compression Ratio Ion Mobility Programming (CRIMP) Accumulation and Compression of Billions of Ions for Ion Mobility-Mass Spectrometry Using Traveling Waves in Structures for Lossless Ion Manipulations (SLIM).

We report on the implementation of a traveling wave (TW) based compression ratio ion mobility programming (CRIMP) approach within structures for lossless ion manipulations (SLIM) that enables both greatly enlarged trapped ion charge capacities and also efficient ion population compression for use in ion mobility (IM) separations. Ion accumulation is conducted in a SLIM serpentine ultralong path with extended routing (SUPER) region after which CRIMP compression allows the large ion populations to be "squeezed". The SLIM SUPER IM module has two regions, one operating with conventional traveling waves (i.e., traveling trap; TT region) and the second having an intermittently pausing or "stuttering" TW (i.e., stuttering trap; ST region). When a stationary voltage profile was used in the ST region, ions are blocked at the TT-ST interface and accumulated in the TT region and then can be released by resuming a conventional TW in the ST region. The population can also be compressed using CRIMP by the repetitive merging of ions distributed over multiple TW bins in the TT region into a single TW bin in the ST region. Ion accumulation followed by CRIMP compression provides the basis for the use of larger ion populations for IM separations. We show that over 109 ions can be accumulated with high efficiency in the present device and that the extent of subsequent compression is only limited by the space charge capacity of the trapping region. Approximately 5 × 109 charges introduced from an electrospray ionization source were trapped for a 40 s accumulation period, more than 2 orders of magnitude greater than the previously reported charge capacity of an ion funnel trap. Importantly, we show that extended ion accumulation in conjunction with CRIMP compression and multiple passes through the serpentine path provides the basis for a highly desirable combination of ultrahigh sensitivity and SLIM SUPER high-resolution IM separations.

Gas phase hydration of halogenated benzene cations. Is it hydrogen or halogen bonding?

Halogen bonding (XB) non-covalent interactions can be observed in compounds containing chlorine, bromine, or iodine which can form directed close contacts of the type R1-XY-R2, where the halogen X acts as a Lewis acid and Y can be any electron donor moiety including electron lone pairs on hetero atoms such as O and N, or π electrons in olefin double bonds and aromatic conjugated systems. In this work, we present the first evidence for the formation of ionic halogen bonds (IXBs) in the hydration of bromobenzene and iodobenzene radical cations in the gas phase. We present a combined thermochemical investigation using the mass-selected ion mobility (MSIM) technique and density functional theory (DFT) calculations of the stepwise hydration of the fluoro, chloro, bromo, and iodobenzene radical cations. The binding energy associated with the formation of an IXB in the hydration of the iodobenzene cation (11.2 kcal mol-1) is about 20% higher than the typical unconventional ionic hydrogen bond (IHB) of the CHδ+OH2 interaction. The formation of an IXB in the hydration of the iodobenzene cation involves a significant entropy loss (29 cal mol-1 K-1) resulting from the formation of a more ordered structure and a highly directional interaction between the oxygen lone pair of electrons of water and the electropositive region around the iodine atom of the iodobenzene cation. In comparison, the hydration of the fluorobenzene and chlorobenzene cations where IHBs are formed, -ΔS° = 18-21 cal mol-1 K-1 consistent with the formation of less ordered structures and loose interactions. The electrostatic potentials on the lowest energy structures of the hydrated halogenated benzene radical cations show clearly that the formation of an IXB is driven by a positively charged σ-hole on the external side of the halogen atom X along the C-X bond axis. The size of the σ-hole increases significantly in bromobenzene and iodobenzene radical cations which results in strong interaction potentials with the electron lone pairs of the oxygen atom of the water molecules and thus IXBs provide the most stable hydrated structures of the bromobenzene and iodobenzene radical cations. The results clearly distinguish the hydration behaviors resulting from the ionic hydrogen and halogen bonding interactions of fluorobenzene and iodobenzene cations, respectively, and establish the different bonding and structural features of the two interactions.

Unconventional CH(δ+)···N hydrogen bonding interactions in the stepwise solvation of the naphthalene radical cation by hydrogen cyanide and acetonitrile molecules.

Equilibrium thermochemical measurements using the mass-selected ion mobility (MSIM) technique have been utilized to investigate the binding energies and entropy changes of the stepwise association of hydrogen cyanide (HCN) and acetonitrile (CH3CN) molecules with the naphthalene radical cation (C10H8˙(+)) in the gas phase forming the C10H8˙(+)(HCN)n and C10H8˙(+)(CH3CN)n clusters with n = 1-3 and 1-5, respectively. The lowest energy structures of the C10H8˙(+)(HCN)n and C10H8˙(+)(CH3CN)n clusters for n = 1-2 have been calculated using the M062X and ω97XD methods within the 6-311+G** basis set, and for n = 1-6 using the B3LYP method within the 6-311++G** basis set. In both systems, the initial interaction occurs through unconventional CH(δ+)···N ionic hydrogen bonds between the hydrogen atoms of the naphthalene cation and the lone pair of electrons on the N atom of the HCN or the CH3CN molecule. The binding energy of CH3CN to the naphthalene cation (11 kcal mol(-1)) is larger than that of HCN (7 kcal mol(-1)) due to a stronger ion-dipole interaction resulting from the large dipole moment of CH3CN (3.9 D). On the other hand, HCN can form both unconventional hydrogen bonds with the hydrogen atoms of the naphthalene cation (CH(δ+)···NCH), and conventional linear hydrogen bonding chains involving HCN···HCN interactions among the associated HCN molecules. HCN molecules tend to form "externally solvated" structures with the naphthalene cation where the naphthalene ion is hydrogen bonded to the exterior of an HCN···HCN chain. For the C10H8˙(+)(CH3CN)n clusters, "internally solvated" structures are favored where the acetonitrile molecules are directly interacting with the naphthalene cation through CH(δ+)···N unconventional ionic hydrogen bonds. In both the C10H8˙(+)(HCN)n and C10H8˙(+)(CH3CN)n clusters, the sequential binding energy decreases stepwise to about 6-7 kcal mol(-1) by three HCN or CH3CN molecules, approaching the macroscopic enthalpy of vaporization of liquid HCN (6.0 kcal mol(-1)).

Communication: Ion mobility of the radical cation dimers: (Naphthalene)2(+•) and naphthalene(+•)-benzene: Evidence for stacked sandwich and T-shape structures.

Dimer radical cations of aromatic and polycyclic aromatic molecules are good model systems for a fundamental understanding of photoconductivity and ferromagnetism in organic materials which depend on the degree of charge delocalization. The structures of the dimer radical cations are difficult to determine theoretically since the potential energy surface is often very flat with multiple shallow minima representing two major classes of isomers adopting the stacked parallel or the T-shape structure. We present experimental results, based on mass-selected ion mobility measurements, on the gas phase structures of the naphthalene(+⋅) ⋅ naphthalene homodimer and the naphthalene(+⋅) ⋅ benzene heterodimer radical cations at different temperatures. Ion mobility studies reveal a persistence of the stacked parallel structure of the naphthalene(+⋅) ⋅ naphthalene homodimer in the temperature range 230-300 K. On the other hand, the results reveal that the naphthalene(+⋅) ⋅ benzene heterodimer is able to exhibit both the stacked parallel and T-shape structural isomers depending on the experimental conditions. Exploitation of the unique structural motifs among charged homo- and heteroaromatic-aromatic interactions may lead to new opportunities for molecular design and recognition involving charged aromatic systems.

Formation of covalently bonded polycyclic hydrocarbon ions by intracluster polymerization of ionized ethynylbenzene clusters.

Here we report a detailed study aimed at elucidating the mechanism of intracluster ionic polymerization following the electron impact ionization of van der Waals clusters of ethynylbenzene (C8H6)n generated by a supersonic beam expansion. The structures of the C16H12, C24H18, C32H24, C40H30, and C48H36 radical cations resulting from the intracluster ion-molecule addition reactions have been investigated using a combination of mass-selected ion dissociation and ion mobility measurements coupled with theoretical calculations. Noncovalent structures can be totally excluded primarily because the measured fragmentations cannot result from noncovalent structures, and partially because of the large difference between the measured collision cross sections and the calculated values corresponding to noncovalent ion-neutral complexes. All the mass-selected cluster ions show characteristic fragmentations of covalently bonded molecular ions by the loss of stable neutral fragments such as CH3, C2H, C6H5, and C7H7. The population of the C16H12 dimer ions is dominated by structural isomers of the type (C6H5)-C≡C-CH(•+)CH-(C6H5), which can grow by the sequential addition of ethynylbenzene molecules, in addition to some contributions from cyclic isomers such as the 1,3- or 1,4-diphenyl cyclobutadiene ions. Similarly, two major covalent isomers have been identified for the C24H18 trimer ions: one that has a blocked cyclic structure assigned to 1,2,4- or 1,3,5-triphenylbenzene cation, and a second isomer of the type (C6H5)-C≡C-C(C6H5)═CH-CH(•+)CH-(C6H5) where the covalent addition of further ethynylbenzene molecules can occur. For the larger ions such as C32H24, C40H30, and C48H36, the major isomers present involve the growing oligomer sequence (C6H5)-C≡C-[C(C6H5)═CH]n-CH(•+)CH-(C6H5) with different locations and orientations of the phenyl groups along the chain. In addition, the larger ions contain another family of structures consisting of neutral ethynylbenzene molecules associated with the blocked cyclic isomer ions such as the diphenylcyclobutadiene and triphenylbenzene cations. Low-energy dissociation channels corresponding to evaporation of ethynylbenzene molecules weakly associated with the covalent ions are observed in the large clusters in addition to the high-energy channels corresponding to fragmentation of the covalently bonded ions. However, in small clusters only high-energy dissociation channels are observed corresponding to the characteristic fragmentation of the molecular ions, thus providing structural signatures to identify the product ions and establish the mechanism of intracluster ionic polymerization.

Proton-bound dimers of nitrogen heterocyclic molecules: substituent effects on the structures and binding energies of homodimers of diazine, triazine, and fluoropyridine.

The bonding energies of proton-bound homodimers BH(+)B were measured by ion mobility equilibrium studies and calculated at the DFT B3LYP/6-311++G** level, for a series of nitrogen heterocyclic molecules (B) with electron-withdrawing in-ring N and on-ring F substituents. The binding energies (ΔH°(dissoc)) of the proton-bound dimers (BH(+)B) vary significantly, from 29.7 to 18.1 kcal/mol, decreasing linearly with decreasing the proton affinity of the monomer (B). This trend differs significantly from the constant binding energies of most homodimers of other organic nitrogen and oxygen bases. The experimentally measured ΔH°(dissoc) for (1,3-diazine)2H(+), i.e., (pyrimidine)2H(+) and (3-F-pyridine)2H(+) are 22.7 and 23.0 kcal/mol, respectively. The measured ΔH°(dissoc) for the pyrimidine(·+)(3-F-pyridine) radical cation dimer (19.2 kcal/mol) is signifcantly lower than that of the proton-bound homodimers of pyrimidine and 3-F-pyridine, reflecting the stronger interaction in the ionic H-bond of the protonated dimers. The calculated binding energies for (1,2-diazine)2H(+), (pyridine)2H(+), (2-F-pyridine)2H(+), (3-F-pyridine)2H(+), (2,6-di-F-pyridine)2H(+), (4-F-pyridine)2H(+), (1,3-diazine)2H(+), (1,4-diazine)2H(+), (1,3,5-triazine)2H(+), and (pentafluoropyridine)2H(+) are 29.7, 24.9, 24.8, 23.3, 23.2, 23.0, 22.4, 21.9, 19.3, and 18.1 kcal/mol, respectively. The electron-withdrawing substituents form internal dipoles whose electrostatic interactions contribute to both the decreased proton affinities of (B) and the decreased binding energies of the protonated dimers BH(+)B. The bonding energies also vary with rotation about the hydrogen bond, and they decrease in rotamers where the internal dipoles of the components are aligned efficiently for inter-ring repulsion. For compounds substituted at the 3 or 4 (meta or para) positions, the lowest energy rotamers are T-shaped with the planes of the two rings rotated by 90° about the hydrogen bond, while the planar rotamers are weakened by repulsion between the ortho hydrogen atoms of the two rings. Conversely, in ortho-substituted (1,2-diazine)2H(+) and (2-F-pyridine)2H(+), attractive interactions between the ortho (C-H) hydrogen atoms of one ring and the electronegative ortho atoms (N or F) of the other ring are stabilizing, and increase the protonated dimer binding energies by up to 4 kcal/mol. In all of the dimers, rotation about the hydrogen bond can involve a 2-4 kcal/mol barrier due to the relative energies of the rotamers.

Ion Mobility Separations Using Cocentric Architecture.

Ion mobility separations, especially using drift tube ion mobility spectrometers, are usually performed in linear channels, which can have a large footprint when extended to achieve higher resolving powers. In this work, we explored the performance of an ion mobility device with a curved architecture, which can have a more compact form. The cocentric ion mobility spectrometer (CoCIMS) manipulates ions between two cocentric surfaces containing a serpentine track. The mobility separation inside the CoCIMS is achieved using traveling waveforms (TWs). We initially evaluated the device using ion trajectory simulations using SIMION, which indicated that when ions traveled circularly inside the CoCIMS they resulted in similar resolving powers and transmitted m/z range as traveling in a straight path. We then performed experimental validation of the CoCIMS in conjunction with a TOF MS. The CoCIMS was made of two flexible printed circuit board materials folded into cocentric cylinders separated by a gap of 2.8 mm. The device was about 50 mm diameter ×152 mm long and provided 1.846 m of serpentine path length. Three sets of mixtures (Agilent tune mixture, tetraalkylammonium salts, and an eight-peptide mixture) and four traveling waveform profiles (square, sine, triangle, and sawtooth) were used. The sawtooth TW profile produced a slightly higher resolving power for the Agilent tuning mixture and tetraalkylammonium ions. The average resolving power for Agilent tune mixture ions ranged from 37 (using sawtooth TW) to 27 (using square TW). The average resolving powers ranged from 45 (sawtooth TW) to 31 (square TW) for tetraalkylammonium ions. The resolving power of the peptide mixture ions was similar among the four TW profiles and ranged from 51 to 56. The average percent error in TWCCS for the peptide mixture ions was about 0.4%. The new device showed promising results, but improvements are needed to further increase the resolving power.

Accumulation of Large Ion Populations with High Ion Densities and Effects Due to Space Charge in Traveling Wave-Based Structures for Lossless Ion Manipulations (SLIM) IMS-MS.

The accumulation of very large ion populations in traveling wave (TW)-based Structures for Lossless ion Manipulations (SLIM) has been studied to better understand aspects of "in-SLIM" ion accumulation, and particularly its use in conjunction with ion mobility spectrometry (IMS). A linear SLIM ion path was implemented that had a "gate" for blocking and accumulating ions for arbitrary time periods. Removing the gate potential caused ions to exit, and the spatial distributions of accumulated ions examined. The ion populations for a set of peptides increased approximately linearly with increased accumulation times until space change effects became significant, after which the peptide precursor ion populations decreased due to growing space charge-related ion activation, reactions, and losses. Ion activation increased with added storage times and the TW amplitude. Lower amplitude TWs in the accumulation/storage region prevented or minimized ion losses or ion heating effects that can also lead to fragmentation. Our results supported the use of an accumulation region close to the SLIM entrance for speeding accumulation, minimizing ion heating, and avoiding ion population profiles that result in IMS peak tailing. Importantly, space charge-driven separations were observed for large populations of accumulated species and attributed to the opposing effects of space charge and the TW. In these separations, ion species form distributions or peaks, sometimes moving against the TW, and are ordered in the SLIM based on their mobilities. Only the highest mobility ions located closest to the gate in the trapped ion population (and where the highest ion densities were achieved) were significantly activated. The observed separations may offer utility for ion prefractionation of ions and increasing the dynamic range measurements, increasing the resolving power of IMS separations by decreasing peak widths for accumulated ion populations, and other purposes benefiting from separations of extremely large ion populations.