Ion mobility spectrometer / mass spectrometer (IMS-MS).

The 1, 2-unsaturated derivatives of (1-4) linked disaccharides serve as versatile building blocks for synthesizing biologically active compounds. Distinguishing between four pairs of stereoisomers in mixtures presents a challenging task. In this study, disaccharide derivatives are analyzed as alkali metal adducts using electrospray ionization tandem mass spectrometry (ESI-MS/MS), both as individual compounds and in mixtures by ion mobility mass spectrometry (IMS). Electrospray ionization (ESI) in combination with tandem mass spectrometry (MS/MS) in positive mode was employed to differentiate lithium adduct ions of hexa-acetyl/hexa-benzyl-D-lactals, and hexa-acetyl/hexa-benzyl-D-maltals, along with their corresponding 2-C-branched malonates. The high resolving power of trapped ion mobility spectrometry (TIMS) with the imeX™ functionality rapidly identified different metal adducts (Li, Na, Cs) as individual isomers and separated mixtures of stereoisomers. The measured collisional cross section (CCS) values were analyzed in relation to predicted CCS values. MS/MS spectra of the [M + Li]+ ions of glycal disaccharide analogues exhibited typical cross-ring and glycosidic bond cleavages. Collision-induced dissociation (CID) spectra provided insights into their fragmentation behavior, allowing differentiation of (1-4) linked disaccharides. TIMS technology delivered adjustable ion mobility resolution for suitable separation of the four sets of stereo isomeric compounds. However, accurately predicting CCS values to differentiate between respective isomeric pairs using the SigmaCCS program for sodium adducts is only partially achievable. ESI CID spectra of [M + Li]+ adduct ions for individual glycal disaccharide analogues facilitate the discrimination between alpha and beta (1-4) linked unsaturated disaccharides and their 2-C-branched analogues. Through optimized experimental conditions, complete baseline ion mobility separation of stereoisomer pairs of the Cs adducts is achieved.

Rationale Saponins are natural compounds presenting a high structural diversity whose structural characterization remains extremely challenging. Ideally, saponin structures are best established using nuclear magnetic resonance experiments conducted on isolated molecules. However, saponins are also increasingly characterized using tandem mass spectrometry (MS/MS) coupled with liquid chromatography, even if collision‐induced dissociation (CID) experiments are often quite limited in accurately determining the saponin structure. Methods We consider here ion mobility mass spectrometry (IMMS) as an orthogonal tool for the structural characterization of saponin isomers by comparing the experimental collisional cross sections (CCSs) of saponin ions with theoretical CCSs for candidate ion structures. Indeed, state‐of‐the‐art theoretical calculations perfectly complement the experimental results, allowing the ion structures to be deciphered at the molecular level. Results We demonstrate that ion mobility can contribute to the structural characterization of saponins because different saponin ions present significantly distinct CCSs. Depending on the nature of the cation (in the positive ion mode), the differences in CCSs can also be exacerbated, optimizing the gas‐phase separation. When associated with molecular dynamics simulations, the CCS data can be used to describe the interactions between the cations, i.e. H + , Na + and K + , and the saponin molecules at a molecular level. Conclusions Our work contributes to resolve the relationship between the primary and secondary structures of saponin ions. However, it is obvious that the structural diversity and complexity of the saponins cannot be definitively unraveled by measuring a single numerical value, here the CCS. Consequently, the structural characterization of unknown saponins will be difficult to achieve based on IMMS alone. Nevertheless, we demonstrated that monodesmosidic and bidesmosidic saponins can be distinguished via IMMS.

Enhancing Biological LC-MS Analyses Using Ion Mobility Spectrometry

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

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.

The detection of illicit explosives in a large room, container, or cargo hold is problematic due to the current limitations in sampling and detection of explosives and to the size and time constraints of the search. Solid Phase MicroExtraction (SPME) can be used to rapidly extract volatile and semi-volatile compounds from the headspace of an explosive and ion mobility spectrometry (IMS) is a rapid presumptive organic detection technique that has already found widespread use in the detection of hidden explosives. SPME has recently been coupled to IMS as a sample pre-concentration device in order to improve the detection of explosives concealed in open areas. Detection limits have been determined for the following taggants: 2-nitrotoluene (2-NT), 4-nitrotoluene (4-NT), 2,3-dimethyl-2,3-dinitro butane (DMNB) and the following volatile explosive compounds: 2,4-dinitrotoluene, (2,4-DNT), 2,6-dinitrotoluene (2,6-DNT), and 2,4,6-trinitrotoluene (2,4,6-TNT). Nitrocellulose (NC) is also capable of being detected by the SPME-IMS system on a reliable basis. Results from these experiments point towards the usefulness of this technique as a potential screening tool for explosive compounds. Mass transport experiments are being conducted to determine the compound concentration in a flow of air for detection to occur. Further work will also be conducted using explosive odor signature compounds as potential illicit explosive detection compounds.

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.

An ion mobility quadrupole time-of-flight mass spectrometry-based pesticide suspect screening methodology was developed and validated covering 20 plant-derived food matrices deriving from six commodity groups of different complexity according to the actual European Commission document SANTE/11813/2017 applying a QuEChERS sample preparation protocol. The method combines ultra-performance liquid chromatography, traveling wave ion mobility, and quadrupole time-of-flight mass spectrometry. Besides the determination of the physicochemical property collision cross-section and the establishment of a corresponding scientific suspect screening database comprising 280 pesticides for several pesticides, different protomers, sodium adducts, as well as dimers were identified in ion mobility spectrometry traces. Additionally, collision cross-section values were included in the validation requirements regarding chromatography and mass spectrometry for the detection of pesticides. A collision cross-section value window was analyzed within a tolerable error of ±2%. For this cross-matrix validation, screening detection limits were determined at concentration levels of 0.100mg/kg (84% of the original pesticide scope), 0.010mg/kg (56%), and 0.001mg/kg (21%). By application of ion mobility spectrometry, the compound identification was improved due to independence of commodity of concern and concentration levels of analyte molecules, as false assignments are reduced by application of a collision cross-section range.

Chapter 5 - High-Performance Ion Mobility Spectrometry

Amyloid structures accumulate and propagate through self-assembly of partially folded proteins and peptides, resulting in a range of disease states. Key to understanding amyloid disease is the characterisation of the often toxic oligomeric species formed during the early stages of fibril assembly. Electrospray ionisation- ion mobility spectrometry - mass spectrometry (ESI-IMS-MS) has emerged as a powerful tool to investigate amyloid oligomer assembly and protein conformation change. In this review we focus on the role of ESI-IMS-MS in understanding and probing conformational changes and the early stages of protein aggregation.

The Paternò-Büchi (PB) reaction is a common organic reaction in which a carbonyl radical formed by exposure to UV radiation reacts with an alkene to form an oxetane ring. Recent analytical applications of this reaction have included the determination of C═C bond position in lipid fatty acyl tails using tandem mass spectrometry. Our group has recently investigated methods for structurally modifying steroid isomers to improve their identification and resolution using ion mobility spectrometry. Herein, we report the first application of the Paternò-Büchi reaction to form steroid oxetanes using a simple, low-cost, and high efficiency method with a low pressure mercury lamp. This methodology is performed on several endogenous steroid isomers, resulting in unique ion mobility spectra that provide a unique fingerprint for each. These fingerprint spectra can add confidence in identification of those compounds, especially in complex biological matrixes. Testosterone and epitestosterone, an epimer pair commonly interrogated in a number of applications such as for their use as performance enhancing drugs, displayed one and three unique ion mobility peaks, respectively. These spectra and their measured collision cross sections (CCS) allow for unambiguous differentiation of these and several other steroid isomer groups analyzed in this work. Finally, multiple anabolic androgenic steroids prohibited by the World Anti-Doping Agency were tested with this method and resulted in unique CCS for their PB reaction products. This approach can offer improved confidence in their identification as well as for many other banned substances.

A critical review of ion mobility spectrometry for the detection of explosives and explosive related compounds

Chondroitin sulfate (CS) glycosaminoglycans are bioactive sulfated polysaccharides comprising repeating units of uronic acid and N-acetyl galactose sulfated at various positions. The optimal length and sulfation pattern of the CS bioactive sequences remain elusive so that structure-activity relationships cannot be easily established. Development of efficient analytical methods allowing the differentiation of the various sulfation patterns of CS sequences is therefore of particular importance to correlate their biological functions to the sulfation pattern. Discrimination of different oligomers (dp2 to dp6) of synthetic chondroitin sulfate isomers was evaluated by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in the negative-ion mode from deprotonated and alkali adduct species. In addition, ion mobility mass spectrometry (IMS-MS) was used to study the influence of both the degree of polymerization and sulfate group location on the gas-phase conformation of CS oligomers. ESI-MS/MS spectra of chondroitin sulfate isomers show characteristic product ions exclusively from alkali adduct species (Li, Na, K and Cs). Whatever the alkali adducts studied, MS/MS of chondroitin oligosaccharides sulfated at position 6 yields a specific product ion at m/z 139 while CS oligosaccharides sulfated at position 4 show a specific product ion at m/z 154. Being observed for the different CS oligomers di-, tetra- and hexasaccharides, these fragment ions are considered as diagnostic ions for chondroitin 6-O-sulfate and chondroitin 4-O-sulfate, respectively. IMS-MS experiments reveal that collision cross-sections (CCS) of CS oligomers with low charge states evolved linearly with degrees of polymerization indicating a similar gas-phase conformation. This study allows the fast and unambiguous differentiation of CS isomers sulfated at position 6 or 4 for both saturated and unsaturated analogues from MS/MS experiments. In addition, the CCS linear evolution of CS oligomers in function of the degree of polymerization indicates that no folding occurs even for hexasaccharides.

Lipid A, a crucialcomponent of lipopolysaccharides (LPS), playsa pivotal role in the pathogenesis of Gram-negative bacteria. LipidA patterns are recognized by mammals and can induce immunostimulatoryeffects. However, the outcome of the interaction is highly dependenton the chemical composition of individual lipid A species. The diversityof potential fatty acyl and polar headgroup combinations in this complexsaccharolipid presents a significant analytical challenge. Currentmass spectrometry (MS)-based lipid A methods are focused on eitherdirect matrix-assisted laser desorption/ionization (MALDI)-MS screeningor comprehensive structural elucidation by tandem mass spectrometry(MS/MS) hyphenated with separation techniques. In this study, we developedan alternative workflow for rapid lipid A profiling covering the entireanalysis pipeline from sample preparation to data analysis. This workflowis based on microextraction and subsequent MALDI-MS/MS analysis ofuropathogenic Escherichia coli utilizingtrapped ion mobility spectrometry (TIMS), followed by mzmine dataprocessing. The additional TIMS dimension served for enhanced sensitivity,selectivity, and structural elucidation through mobility-resolvedfragmentation via parallel accumulation-serial fragmentation (PASEF)in parallel reaction monitoring (prm)-mode. Furthermore, mzmine enabledautomated MS/MS acquisition by adapting the spatial ion mobility-scheduledexhaustive fragmentation (SIMSEF) strategy for MALDI spot analysis.It also facilitated robust lipid A annotation through a newly developedextension of the rule-based lipid annotation module, allowing forthe custom generation of lipid classes, including specific fragmentationrules. In this study, the first publication of lipid A species’collision cross section (CCS) values is reported, which will enhancehigh-confidence lipid A annotation in future studies.

A multidimensional analytical approach based on time-decoupled online comprehensive two-dimensional liquid chromatography coupled with ion mobility quadrupole time-of-flight mass spectrometry for the analysis of ginsenosides from white and red ginsengs