Enhancing Biological LC-MS Analyses Using Ion Mobility Spectrometry

Advances in LC-MS strategies for comprehensive characterization and quantification of antibody-drug conjugates in preclinical and clinical settings.

As an effective and efficient analytical device ion mobility spectrometry (IMS) is routinely used in many critical trace detection applications. The role of IMS is steadily realized in many emerging applications as well, especially in biological/medical sciences, for example, in exploration of complex metabolomics, structural proteomics, etc. Increasing accuracy of mobility values, manipulating drift medium and reaction kinetics, enhancing sensitivity both algorithmically and by improved electronics, and above all, miniaturization with high performance, form some of the key topics for advancing the science and technology of IMS. New problems continue rejuvenating researchers and engineers to identify key questions, derive answers through systematic scientific studies and advance the boundary of technological development. While different configurations of this mobility-based device have been realized as a stand-alone technology, these devices are interfaced or hyphenated with other orthogonal sensing modalities for much enhanced performance. In all of these explorations modeling and simulation (M&S) and theoretical work act as an underpinning tool to develop new design concepts, build their operational paradigms, and optimize performance metrics. It is well recognized that IMS operation includes several distinct steps. Broadly speaking, ionization, pulsing/ gating, ion transport, ion detection, and algorithm for data exploitation constitute the key elements of IMS operation. While significant progress has been made in answering many questions, the complexity of each of these problems is exacerbated due to an overarching need of miniaturization without compromising performance metrics. Some of the key questions pertain to the mechanism of the reactant and the product ion formation for different ionization processes, effective gating and injection of charges from the ionization to drift region, ion-ion and ion-neutral interactions, ion diffusion, coulomb interactions and charge control, induced charge, screening of Faraday plate, and amplifier characteristics. Critical problems in these areas are being addressed by mobilizing expertise from all domains of science and engineering as well as in mathematics and statistics. In this vein, modeling and simulation, if sufficiently validated, can be very effectively utilized to gain insight into the problems and obtain guidance to design experiments. In further advancing the utility of the technology it will be important to undertake a modeling and simulation task integrating the end-toend process steps which will help building the overall system link budget. Accurately capturing the phenomenology governing each step that entails acquiring samples from a target, ionizing and transporting through IMS and finally detecting analyte ions will form the crux of the effort. While this special issue by no means encompasses the entire span of modeling and simulation (M&S) activities in IMS, each article presents a scholarly approach to problems of significant current interest and demonstrates the merit of M&S. This special issue is offered to the community with the hope that it will provide a compendium sampling some of the areas of current interests on IMS modeling and simulation. * Samar K. Guharay sguharay@mitre.org

Different environmental conditions in which, equipment based on differential ion mobility spectrometry must operate, forced designers and engineers to analyze the impact of, among other things, external factors on their performance and efficiency. These devices, thanks to the DMS technology, can identify and characterize the ions contained in the gaseous state. However, many areas of this technique remain undiscovered, which should be examined in more detail. One such aspect is the influence of the temperature of gaseous medium, containing tested analyte. The research presented in this article shows why temperature is an important factor on the distribution of generated peaks. The results are shown for different spectra generated carrier gas temperatures (50°C - 80°C) in which ions were toxic warfare agents. Based on those graphs, it can be stated that increase of the temperature allows for better separation of the peaks from the background. Because of the similar ion mobility of the analyte and background ions for high and low electric field, DMS device can send false alarms, due to the poor interpretations of passing a signal to them. So to be able to accurately assess the level of risk due to the presence of substances BST in air, the test medium was added to isobutanol and isopropanol additives. They help better analyze and separate measured ions.

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

Rechargeable aluminum–sulfur (Al–S) batteries are considered promising candidates for next-generation energy storage owing to the natural abundance, intrinsic safety, low cost, and high gravimetric capacities of aluminum and sulfur (2980 mAh g⁻¹ and 1675 mAh g⁻¹, respectively). However, their practical deployment has been hindered by the scarcity of electrolytes that can simultaneously support reversible aluminum electrodeposition and sulfur electroreduction, while also minimizing polarization losses. Alkali chloroaluminate molten salts (AlCl₃–NaCl–KCl) were recently used as electrolytes for Al–S batteries instead of conventional AlCl₃–EMImCl (1-ethyl-3-methylimidazolium chloride) ionic liquids, demonstrating significantly reduced polarization at elevated temperature (e.g., 110 °C), though its origins are poorly understood.In this work, we investigate how electrolyte speciation, ion mobility, and soluble intermediates in these two electrolyte systems influence polarization losses in rechargeable Al–S batteries. Variable-temperature liquid-state 27Al, 23Na, and 1H single-pulse and relaxation nuclear magnetic resonance (NMR) measurements were conducted under quantitative conditions to probe electrolyte speciation and ion dynamics. 23Na and 1H pulsed-field-gradient (PFG) NMR experiments were also performed to quantify cation diffusion coefficients and their activation energies for diffusion. Additionally, integrated 27Al and 23Na NMR signal intensities of the molten salt electrolyte enabled accurate measurements of its freezing and melting temperature range, which coupled with changes in ion mobility, yielded insights into the optimum operating temperature. Differential scanning calorimetry (DSC) was also performed to measure these thermodynamic phase transitions and analyzed with respect to the NMR data. Solid-state 27Al and 23Na single-pulse NMR measurements of both pristine and cycled molten salt electrolytes provided mechanistic insights into the electrochemical conversion reaction, including the formation of electrolyte soluble polysulfide-like intermediates. In addition, we formulated quaternary electrolyte mixtures comprising AlCl₃, NaCl, KCl, and a fourth component (e.g., [EMIm]Cl, urea, or LiCl) with the objective of lowering the operating temperature and understanding the effects on cell polarization. Al-S batteries were prepared using the different electrolytes to compare their overpotentials, as well as specific capacities, cycle life, and cell impedance. Overall, the roles of electrolyte speciation, ion mobility and soluble intermediates in governing the overpotential in Al-S batteries were elucidated, providing new insights for the rational design of low cost, energy dense electrolytes for Al-S batteries.

Rotationally averaged collision cross section (CCS) values for a series of proteins and protein complexes ranging in size from 8.6 to 810 kDa are reported. The CCSs were obtained using a native electrospray ionization drift tube ion mobility-Orbitrap mass spectrometer specifically designed to enhance sensitivity while having high-resolution ion mobility and mass capabilities. Periodic focusing (PF)-drift tube (DT)-ion mobility (IM) provides first-principles determination of the CCS of large biomolecules that can then be used as CCS calibrants. The experimental, first-principles CCS values are compared to previously reported experimentally determined and computationally calculated CCS using projected superposition approximation (PSA), the Ion Mobility Projection Approximation Calculation Tool (IMPACT), and Collidoscope. Experimental CCS values are generally in agreement with previously reported CCSs, with values falling within ∼5.5%. In addition, an ion mobility resolution (CCS centroid divided by CCS fwhm) of ∼60 is obtained for pyruvate kinase (MW ∼ 233 kDa); however, ion mobility resolution for bovine serum albumin (MW ∼ 68 kDa) is less than ∼20, which arises from sample impurities and underscores the importance of sample quality. The high resolution afforded by the ion mobility-Orbitrap mass analyzer provides new opportunities to understand the intricate details of protein complexes such as the impact of post-translational modifications (PTMs), stoichiometry, and conformational changes induced by ligand binding.

Since the development of electrospray ionization (ESI) for ion mobility spectrometry mass spectrometry (IMMS), IMMS have been extensively applied for characterization of gas-phase bio-molecules. Conventional ion mobility spectrometry (IMS), defined as drift tube IMS (DT-IMS), is typically a stacked ring design that utilizes a low electric field gradient. Field asymmetric ion mobility spectrometry (FAIMS) is a newer version of IMS, however, the geometry of the system is significantly different than DT-IMS and data are collected using a much higher electric field. Here we report construction of a novel ambient pressure dual gate DT-IMS coupled with a FAIMS system and then coupled to a quadrupole ion trap mass spectrometer (QITMS) to form a hybrid three-dimensional separation instrument, DT-IMS-FAIMS-QITMS. The DT-IMS was operated at ~3 Townsend (electric field/number density (E/N) or (Td)) and was coupled in series with a FAIMS, operated at ~80 Td. Ions were mobility-selected by the dual gate DT-IMS into the FAIMS and from the FAIMS the ions were detected by the QITMS for as either MS or MSn. The system was evaluated using cocaine as an analytical standard and tested for the application of separating three isomeric tri-peptides: tyrosine-glycine-tryptophan (YGW), tryptophan-glycine-tyrosine (WGY) and tyrosine-tryptophan-glycine (YWG). All three tri-peptides were separated in the DT-IMS dimension and each had one mobility peak. The samples were partially separated in the FAIMS dimension but two conformation peaks were detected for the YWG sample while YGW and WGY produced only one peak. Ion validation was achieved for all three samples using QITMS.

Low pressure (<20 Torr) non-thermal plasma flow tube reactors are commonly used for the synthesis of high purity nanoparticles (NPs) via nucleation and subsequent growth from vapor phase precursors. In spite of their utility, process monitoring (i.e. output NP size characterization) in such reactors is difficult, as there is a dearth of techniques available for online NP size distribution function measurement. In this study, we developed and applied an ion mobility spectrometry (IMS) system consisting of a low pressure differential mobility analyzer (LPDMA) and electrical detector to determine the collision cross section distribution functions of Si NPs synthesized in a radio frequency non-thermal SiH4–Ar plasma operated at ∼2 Torr (266 Pa). The collision cross section, roughly proportional to projected area, is a parameter quantifying the size and structure of nanometer scale species in the vapor phase. We introduce the collision cross section distribution function as a metric for potential use in online process monitoring in NP synthesis. Proper inversion of the collision cross section distribution function requires a priori knowledge of the LPDMA transfer function. We utilized a tandem differential mobility analyzer approach, coupled with a Twomey–Markwoski based scheme to determine LPDMA transfer functions. Subsequent application of these transfer functions in collision cross section distribution function determination showed that at the outlet of the non-thermal plasma flow tube reactor, both negatively and positively charged Si NPs persisted with nearly identical collision cross section distribution functions. NPs were found to have mode ‘mobility equivalent’ diameters near 10 nm, and via TEM analysis were found to persist at the reactor outlet as small aggregates composed of ∼5 nm diameter primary particles. Size distribution functions inferred from collision cross section measurements were compared to size distribution functions inferred from TEM images; excellent agreements were found between IMS and TEM for both mean mobility equivalent diameter and distribution function width. In total, this study shows that (1) IMS is a viable approach for process monitoring in non-thermal plasma NP flow tube synthesis systems, (2) although NPs are modestly aggregated at low-pressure plasma reactor outlets, the extent of aggregation is considerably less than observed in most atmospheric pressure and equilibrium NP synthesis systems (e.g. flames), and (3) after exiting the plasma reactor, the decharging of NPs from highly negative charge levels to a bipolar charge distribution likely drives aggregation on the plasma boundary.

High-resolution ion mobility (resolving power > 200) coupled with mass spectrometry (MS) is a powerful analytical tool for resolving isobars and isomers in complex samples. High-resolution ion mobility is capable of discerning additional structurally distinct features, which are not observed with conventional resolving power ion mobility (IM, resolving power ~ 50) techniques such as traveling wave IM and drift tube ion mobility (DTIM). DTIM in particular is considered to be the “gold standard” IM technique since collision cross section (CCS) values are directly obtained through a first-principles relationship, whereas traveling wave IM techniques require an additional calibration strategy to determine accurate CCS values. In this study, we aim to evaluate the separation capabilities of a traveling wave ion mobility structures for lossless ion manipulation platform integrated with mass spectrometry analysis (SLIM IM-MS) for both lipid isomer standards and complex lipid samples. A cross-platform investigation of seven subclass-specific lipid extracts examined by both DTIM-MS and SLIM IM-MS showed additional features were observed for all lipid extracts when examined under high resolving power IM conditions, with the number of CCS-aligned features that resolve into additional peaks from DTIM-MS to SLIM IM-MS analysis varying between 5 and 50%, depending on the specific lipid sub-class investigated. Lipid CCS values are obtained from SLIM IM (TW(SLIM)CCS) through a two-step calibration procedure to align these measurements to within 2% average bias to reference values obtained via DTIM (DTCCS). A total of 225 lipid features from seven lipid extracts are subsequently identified in the high resolving power IM analysis by a combination of accurate mass-to-charge, CCS, retention time, and linear mobility-mass correlations to curate a high-resolution IM lipid structural atlas. These results emphasize the high isomeric complexity present in lipidomic samples and underscore the need for multiple analytical stages of separation operated at high resolution.Graphical abstract

Per- and polyfluoroalkyl substances (PFAS) are a class of thousands of man-made chemicals that are persistent and highly stable in the environment. Fish consumption has been identified as a key route of PFAS exposure for humans. However, routine fish monitoring targets only a handful of PFAS, and non-targeted analyses have largely only evaluated fish from heavily PFAS-impacted waters. Here, we evaluated PFAS in fish fillets from recreational and drinking water sources in central North Carolina to assess whether PFAS are present in these fillets that would not be detected by conventional targeted methods. We used liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) to collect full scan feature data, performed suspect screening using an in-house library of 100 PFAS for high confidence feature identification, searched for additional PFAS features using non-targeted data analyses, and quantified perfluorooctanesulfonic acid (PFOS) in the fillet samples. A total of 36 PFAS were detected in the fish fillets, including 19 that would not be detected using common targeted methods, with a minimum of 6 and a maximum of 22 in individual fish. Median fillet PFOS levels were concerningly high at 11.6 to 42.3 ppb, and no significant correlation between PFOS levels and number of PFAS per fish was observed. Future PFAS monitoring in this region should target more of these 36 PFAS, and other regions not considered heavily PFAS contaminated should consider incorporating non-targeted analyses into ongoing fish monitoring studies.

Ion mobility (IM) is a gas-phase separation technique that is used to determine the collision cross sections of native-like ions of proteins and protein complexes, which are in turn used as restraints for modeling the structures of those analytes in solution. Here, we evaluate the stability of native-like ions using tandem IM experiments implemented using structures for lossless ion manipulations (SLIM). In this implementation of tandem IM, ions undergo a first dimension of IM up to a switch that is used to selectively transmit ions of a desired mobility. Selected ions are accumulated in a trap and then released after a delay to initiate the second dimension of IM. For delays ranging from 16 to 33 231 ms, the collision cross sections of native-like, 7+ cytochrome c ions increase monotonically from 15.1 to 17.1 nm2. The largest products formed in these experiments at near-ambient temperature are still far smaller than those formed in energy-dependent experiments (∼21 nm2). However, the collision cross section increases by ∼2% between delay times of 16 and 211 ms, which may have implications for other IM experiments on these time scales. Finally, two subpopulations from the full population were each mobility selected and analyzed as a function of delay time, showing that the three populations can be differentiated for at least 1 s. Together, these results suggest that elements of native-like structure can have long lifetimes at near-ambient temperature in the gas phase but that gas-phase dynamics should be considered when interpreting results from IM.

Upon development of a workflow to analyze (±)-Verapamil and its metabolites using differential mobility spectrometry (DMS), we noticed that the ionogram of protonated Verapamil consisted of two peaks. This was inconsistent with its metabolites, as each exhibited only a single peak in the respective ionograms. The unique behaviour of Verapamil was attributed to protonation at its tertiary amino moiety, which generated a stereogenic quaternary amine. The introduction of additional chirality upon N-protonation of Verapamil renders four possible stereochemical configurations for the protonated ion: (R,R), (S,S), (R,S), or (S,R). The (R,R)/(S,S) and (R,S)/(S,R) enantiomeric pairs are diastereomeric and thus exhibit unique conformations that are resolvable by linear and differential ion mobility techniques. Protonation-induced chirality appears to be a general phenomenon, as N-protonation of 12 additional chiral amines generated diastereomers that were readily resolved by DMS.

Block-truncated poly(phosphodiester)s are digital macromolecules storing binary information that can be decoded by MS/MS sequencing of individual blocks released as primary fragments of the entire polymer. As such, they are ideal species for the serial sequencing methodology enabled by MS-(CID)-IMS-(CID)-MS coupling, where two activation stages are combined in-line with ion mobility spectrometry (IMS) separation. Yet, implementation of this coupling still requires efforts to achieve IMS resolution of inner blocks, that can be considered as small oligomers with α termination composed of one nitroxide decorated with a different tag. As shown by molecular dynamics simulation, these oligomers adopt a conformation where the tag points out of the coil formed by the chain. Accordingly, the sole nitroxide termination was investigated here as a model to reduce the cost of calculation aimed at predicting the shift of collision cross-section (CCS) induced by new tag candidates and extrapolate this effect to nitroxide-terminated oligomers. A library of 10 nitroxides and 7 oligomers was used to validate our calculation methods by comparison with experimental IMS data as well as our working assumption. Based on conformation predicted by theoretical calculation, three new tag candidates could be proposed to achieve the +40 Å2 CCS shift required to ensure IMS separation of oligomers regardless of their coded sequence.

Here we examine the relationship among resolving power (Rp), resolution (Rpp), and collision cross section (CCS) for compounds analyzed in previous ion mobility (IM) experiments representing a wide variety of instrument platforms and IM techniques. Our previous work indicated these three variables effectively describe and predict separation efficiency for drift tube ion mobility spectrometry experiments. In this work, we seek to determine if our previous findings are a general reflection of IM behavior that can be applied to various instrument platforms and mobility techniques. Results suggest IM distributions are well characterized by a Gaussian model and separation efficiency can be predicted on the basis of the empirical difference in the gas-phase CCS and a CCS-based resolving power definition (CCS/ΔCCS). Notably traveling wave (TWIMS) was found to operate at resolutions substantially higher than a single-peak resolving power suggested. When a CCS-based Rp definition was utilized, TWIMS was found to operate at a resolving power between 40 and 50, confirming the previous observations by Giles and co-workers. After the separation axis (and corresponding resolving power) is converted to cross section space, it is possible to effectively predict separation behavior for all mobility techniques evaluated (i.e., uniform field, trapped ion mobility, traveling wave, cyclic, and overtone instruments) using the equations described in this work. Finally, we are able to establish for the first time that the current state-of-the-art ion mobility separations benchmark at a CCS-based resolving power of >300 that is sufficient to differentiate analyte ions with CCS differences as small as 0.5%.

The lowest energy unimolecular dissociation product channel of protonated tyrosine, [Tyr + H]+, is loss of NH3. The structure of the [Tyr - NH3 + H]+ ion is still debated; past calculations suggest that the global minimum benzyl cation form is only accessible via a relatively high barrier and that a lower energy pathway to formation of the higher-energy phenonium isomer is likely to occur via collision-induced dissociation (CID). To resolve this open question, [Tyr - NH3 + H]+ was studied computationally and experimentally using ion mobility spectrometry, ultraviolet photodissociation (UVPD) spectroscopy, and infrared ion spectroscopy (IRIS). Traveling wave ion mobility spectrometry (TWIMS) yields a collision cross section of ΩN2 = 130.0 ± 1.4 Å2, which compares favorably with computed values of and for the benzyl cation and phenonium products, respectively. Differential mobility spectrometry and mass spectrometry were used to mobility- and mass-select [Tyr + H]+ prior to producing [Tyr - NH3 + H]+via CID and subsequently measuring its UVPD spectrum. Similarly, [Tyr - NH3 + H]+ was produced via CID prior to measuring its IRIS spectrum. The UVPD and IRIS spectra indicate that the phenonium ion is the major product formed via CID.