Jody C. May

Conformational ordering of biomolecules in the gas phase: nitrogen collision cross sections measured on a prototype high resolution drift tube ion mobility-mass spectrometer.

Ion mobility-mass spectrometry measurements which describe the gas-phase scaling of molecular size and mass are of both fundamental and pragmatic utility. Fundamentally, such measurements expand our understanding of intrinsic intramolecular folding forces in the absence of solvent. Practically, reproducible transport properties, such as gas-phase collision cross-section (CCS), are analytically useful metrics for identification and characterization purposes. Here, we report 594 CCS values obtained in nitrogen drift gas on an electrostatic drift tube ion mobility-mass spectrometry (IM-MS) instrument. The instrument platform is a newly developed prototype incorporating a uniform-field drift tube bracketed by electrodynamic ion funnels and coupled to a high resolution quadrupole time-of-flight mass spectrometer. The CCS values reported here are of high experimental precision (±0.5% or better) and represent four chemically distinct classes of molecules (quaternary ammonium salts, lipids, peptides, and carbohydrates), which enables structural comparisons to be made between molecules of different chemical compositions for the rapid “omni-omic” characterization of complex biological samples. Comparisons made between helium and nitrogen-derived CCS measurements demonstrate that nitrogen CCS values are systematically larger than helium values; however, general separation trends between chemical classes are retained regardless of the drift gas. These results underscore that, for the highest CCS accuracy, care must be exercised when utilizing helium-derived CCS values to calibrate measurements obtained in nitrogen, as is the common practice in the field.

Ion Mobility-Mass Spectrometry: Time-Dispersive Instrumentation

The field of ion mobility-mass spectrometry (IM-MS) has grown with significant momentum in recent years in both fundamental advances and pioneering applications. A search of the terms “ion mobility” and “mass spectrometry” returns more than 2 000 papers, with over half of these being published in the past 4 years (Figure ​(Figure1,1, left). This increased interest has been motivated in large part by improved technologies which have enabled contemporary IM-MS to be amendable to a variety of samples in biology and medicine with high sensitivity, resolving power, and sample throughput. Figure 1 (Left) Histogram of the number of publications published per year in ion mobility and ion mobility-mass spectrometry. Note that the scale is truncated at 300 to highlight the number of publications specifically utilizing IM-MS. Further distinction is ... Highlights of the historical development of the field are presented in Figure ​Figure1,1, right. Ion mobility and mass spectrometry trace their foundations to the X-ray experiments of Thomson and Rutherford in the late 1800s,1 with Tyndall making significant improvements in the analytical capabilities of ion mobility around the 1930s.2,3 During this early era of discovery, a variety of ion mobility experimental parameters were explored, including differences in pressure,4,5 temperature,6,7 electric field,8 and the ion residence time (age) in the drift region.9 Hybrid IM-MS instruments of various configurations were developed by several groups in the 1960s to study gas-phase ion chemistry.10−12 Ion mobility measurements were used by Dole in the earliest development of electrospray ionization (ESI).13,14 Following commercialization,15 ion mobility instrumentation was used for structure-based characterization16 and differentiation of chemical isomers.17,18 In 1982, laser ionization was demonstrated with ion mobility as a means of generating simplified mobility spectra based on protonated species.19 The features which define modern IM-MS, namely, high resolution, high sensitivity, and broad sample compatibility, were developed in the 1990s and coincided with the rapid development of MS in response to the introduction of ESI and MALDI sample ionization.20−22 The last 2 decades saw significant improvements made in the coupling of IM to MS, notably the use of electrodynamic fields to confine, transfer, and focus ions across disparate pressure regions into high vacuum. An interesting observation to be made in this historical analysis is that many of the features we associate with contemporary ion mobility technology were key aspects of early ion mobility instrument design. Several noteworthy reviews of IM-MS have been published, which cover many aspects of the IM-MS technique and range of applications.23−28 A number of influential books covering various aspects of the ion mobility field are also available.29−34 Of particular relevance is Mason and McDaniel’s Transport Properties of Ions in Gases,35 which was recently republished by the American Society for Mass Spectrometry in their classic books series. Though last revised in 1988, this book is still widely considered the seminal treatment of the motion of ions in gases. The technologies and application areas which IM-MS now encompasses has expanded to such a breadth that new reviews covering IM-MS and related areas now appear every few years in the literature. A comprehensive and critical review of the field as a whole is no longer appropriate nor tractable, and as such it is the intent of this review to focus primarily on recent developments made with regard to temporally dispersive ion mobility techniques (drift tubes and traveling wave separators), with an emphasis on their use specifically in IM-MS instrumentation and methods. This focus is selected because of the recent commercial offerings in this regard that have become widely used in many research environments. The present review is not intended to be comprehensive of the ion mobility advances but rather focuses on time-dispersive IM-MS instrumentation over the past few years.

Neurovascular unit on a chip: implications for translational applications

The blood-brain barrier (BBB) dynamically controls exchange between the brain and the body, but this interaction cannot be studied directly in the intact human brain or sufficiently represented by animal models. Most existing in vitro BBB models do not include neurons and glia with other BBB elements and do not adequately predict drug efficacy and toxicity. Under the National Institutes of Health Microtissue Initiative, we are developing a three-dimensional, multicompartment, organotypic microphysiological system representative of a neurovascular unit of the brain. The neurovascular unit system will serve as a model to study interactions between the central nervous system neurons and the cerebral spinal fluid (CSF) compartment, all coupled to a realistic blood-surrogate supply and venous return system that also incorporates circulating immune cells and the choroid plexus. Hence all three critical brain barriers will be recapitulated: blood-brain, brain-CSF, and blood-CSF. Primary and stem cell-derived human cells will interact with a variety of agents to produce critical chemical communications across the BBB and between brain regions. Cytomegalovirus, a common herpesvirus, will be used as an initial model of infections regulated by the BBB. This novel technological platform, which combines innovative microfluidics, cell culture, analytical instruments, bioinformatics, control theory, neuroscience, and drug discovery, will replicate chemical communication, molecular trafficking, and inflammation in the brain. The platform will enable targeted and clinically relevant nutritional and pharmacologic interventions for or prevention of such chronic diseases as obesity and acute injury such as stroke, and will uncover potential adverse effects of drugs. If successful, this project will produce clinically useful technologies and reveal new insights into how the brain receives, modifies, and is affected by drugs, other neurotropic agents, and diseases.

Evaluation of Collision Cross Section Calibrants for Structural Analysis of Lipids by Traveling Wave Ion Mobility-Mass Spectrometry

Collision cross section (CCS) measurement of lipids using traveling wave ion mobility-mass spectrometry (TWIM-MS) is of high interest to the lipidomics field. However, currently available calibrants for CCS measurement using TWIM are predominantly peptides that display quite different physical properties and gas-phase conformations from lipids, which could lead to large CCS calibration errors for lipids. Here we report the direct CCS measurement of a series of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) in nitrogen using a drift tube ion mobility (DTIM) instrument and an evaluation of the accuracy and reproducibility of PCs and PEs as CCS calibrants for phospholipids against different classes of calibrants, including polyalanine (PolyAla), tetraalkylammonium salts (TAA), and hexakis(fluoroalkoxy)phosphazines (HFAP), in both positive and negative modes in TWIM-MS analysis. We demonstrate that structurally mismatched calibrants lead to larger errors in calibrated CCS values while the structurally matched calibrants, PCs and PEs, gave highly accurate and reproducible CCS values at different traveling wave parameters. Using the lipid calibrants, the majority of the CCS values of several classes of phospholipids measured by TWIM are within 2% error of the CCS values measured by DTIM. The development of phospholipid CCS calibrants will enable high-accuracy structural studies of lipids and add an additional level of validation in the assignment of identifications in untargeted lipidomics experiments.

Ion mobility-mass spectrometry strategies for untargeted systems, synthetic, and chemical biology.

Contemporary strategies that concentrate on only one or a handful of molecular targets limits the utility of the information gained for diagnostic and predictive purposes. Recent advances in the sensitivity, speed, and precision of measurements obtained from ion mobility coupled to mass spectrometry (IM-MS) have accelerated the utility of IM-MS in untargeted, discovery-driven studies in biology. Perhaps most evident is the impact that such wide-scale discovery capabilities have yielded in the areas of systems, synthetic, and chemical biology, where the need for comprehensive, hypothesis-driving studies from multidimensional and unbiased data is required.

Ion Mobility Collision Cross Section Compendium

In this review, we focus on an important aspect of ion mobility (IM) research, namely the reporting of quantitative ion mobility measurements in the form of the gas-phase collision cross section (CCS), which has provided a common basis for comparison across different instrument platforms and offers a unique form of structural information, namely size and shape preferences of analytes in the absence of bulk solvent. This review surveys the over 24,000 CCS values reported from IM methods spanning the era between 1975 to 2015, which provides both a historical and analytical context for the contributions made thus far, as well as insight into the future directions that quantitative ion mobility measurements will have in the analytical sciences. The analysis was conducted in 2016, so CCS values reported in that year are purposely omitted. In another few years, a review of this scope will be intractable, as the number of CCS values which will be reported in the next three to five years is expected to exceed the total amount currently published in the literature.

Advanced Multidimensional Separations in Mass Spectrometry: Navigating the Big Data Deluge

Hybrid analytical instrumentation constructed around mass spectrometry (MS) is becoming the preferred technique for addressing many grand challenges in science and medicine. From the omics sciences to drug discovery and synthetic biology, multidimensional separations based on MS provide the high peak capacity and high measurement throughput necessary to obtain large-scale measurements used to infer systems-level information. In this article, we describe multidimensional MS configurations as technologies that are big data drivers and review some new and emerging strategies for mining information from large-scale datasets. We discuss the information content that can be obtained from individual dimensions, as well as the unique information that can be derived by comparing different levels of data. Finally, we summarize some emerging data visualization strategies that seek to make highly dimensional datasets both accessible and comprehensible.

An Interlaboratory Evaluation of Drift Tube Ion Mobility–Mass Spectrometry Collision Cross Section Measurements

Collision cross section (CCS) measurements resulting from ion mobility-mass spectrometry (IM-MS) experiments provide a promising orthogonal dimension of structural information in MS-based analytical separations. As with any molecular identifier, interlaboratory standardization must precede broad range integration into analytical workflows. In this study, we present a reference drift tube ion mobility mass spectrometer (DTIM-MS) where improvements on the measurement accuracy of experimental parameters influencing IM separations provide standardized drift tube, nitrogen CCS values (DTCCSN2) for over 120 unique ion species with the lowest measurement uncertainty to date. The reproducibility of these DTCCSN2 values are evaluated across three additional laboratories on a commercially available DTIM-MS instrument. The traditional stepped field CCS method performs with a relative standard deviation (RSD) of 0.29% for all ion species across the three additional laboratories. The calibrated single field CCS method, which is compatible with a wide range of chromatographic inlet systems, performs with an average, absolute bias of 0.54% to the standardized stepped field DTCCSN2 values on the reference system. The low RSD and biases observed in this interlaboratory study illustrate the potential of DTIM-MS for providing a molecular identifier for a broad range of discovery based analyses.

The influence of drift gas composition on the separation mechanism in traveling wave ion mobility spectrometry: insight from electrodynamic simulations

The influence of three different drift gases (helium, nitrogen, and argon) on the separation mechanism in traveling wave ion mobility spectrometry is explored through ion trajectory simulations which include considerations for ion diffusion based on kinetic theory and the electrodynamic traveling wave potential. The model developed for this work is an accurate depiction of a second-generation commercial traveling wave instrument. Three ion systems (cocaine, MDMA, and amphetamine) whose reduced mobility values have previously been measured in different drift gases are represented in the simulation model. The simulation results presented here provide a fundamental understanding of the separation mechanism in traveling wave, which is characterized by three regions of ion motion: (1) ions surfing on a single wave, (2) ions exhibiting intermittent roll-over onto subsequent waves, and (3) ions experiencing a steady state roll-over which repeats every few wave cycles. These regions of ion motion are accessed through changes in the gas pressure, wave amplitude, and wave velocity. Resolving power values extracted from simulated arrival times suggest that momentum transfer in helium gas is generally insufficient to access regions (2) and (3) where ion mobility separations occur. Ion mobility separations by traveling wave are predicted to be effectual for both nitrogen and argon, with slightly lower resolving power values observed for argon as a result of band-broadening due to collisional scattering. For the simulation conditions studied here, the resolving power in traveling wave plateaus between regions (2) and (3), with further increases in wave velocity contributing only minor improvements in separations.

Investigation of the Complete Suite of the Leucine and Isoleucine Isomers: Toward Prediction of Ion Mobility Separation Capabilities

In this study we investigated 11 isomers with the molecular formula C6H13NO2 (m/z 131) to ascertain the potential of utilizing drift tube ion mobility mass spectrometry to aid in the separation of isomeric mixtures. This study of small molecules provides a detailed examination of the application of uniform field ion mobility for a narrow scope of isomers with variations in both bond coordination and stereochemistry. For small molecules, it was observed that in general constitutional isomers are more readily separated by uniform field mobility in comparison to stereoisomers such as enantiomers or diastereomers. Diastereomers exhibited differences in their collision cross section (CCS), but were unresolvable in a mixture, whereas the enantiomers studied did not exhibit statistically different CCS values. A mathematical relationship relating the CCS to resolving power was developed in order to predict the required ion mobility resolving power needed to separate the various isomer classes. For the majority of isomers evaluated in this study, a uniform field-based resolving power of 100 was predicted to be sufficient to resolve over half (∼60%) of all hypothetical isomer pairs, including leucine and isoleucine, whereas their stereoisomers (d- and l-forms) are predicted to be significantly more challenging, if not impossible, to separate by conventional drift tube techniques.