Ion Mobility Mass Spectrometry Data Research Articles

Exploring Ion Mobility Mass Spectrometry Data File Conversions to Leverage Existing Tools and Enable New Workflows.

Ion mobility (IM) is often combined with LC-MS experiments to provide an additional dimension of separation for complex sample analysis. While highly complex samples are better characterized by the full dimensionality of LC-IM-MS experiments to uncover new information, downstream data analysis workflows are often not equipped to properly mine the additional IM dimension. For many samples the data acquisition benefits of including IM separations are all that is necessary to uncover sample information and the full dimensionality of the data is not required for data analysis. Postacquisition reduction and adaptation of the dimensions of LC-IM-MS and IM-MS experiments into an LC-MS format opens the possibility to use a plethora of existing software tools. In this work, we developed data file conversion tools to reduce the complexity of IM data analysis. Three data file transformations are introduced in the PNNL PreProcessor software: (1) mapping the IM axis to the LC axis for IM-MS data, (2) converting the drift time vs m/z space to CCS/z vs m/z space, and (3) transforming All Ions IM/MS mobility aligned fragmentation data to a standard LC-MS DDA data file format. These new data file conversions are demonstrated with corresponding lipidomics and proteomics workflows that leverage existing LC-MS data analysis software to highlight the benefits of the data transformations.

Interactions of Curcumin's Degradation Products with the Aβ42 Dimer: A Computational Study.

Amyloid-β (Aβ) dimers are the smallest toxic species along the amyloid-aggregation pathway and among the most populated oligomeric accumulations present in the brain affected by Alzheimer's disease (AD). A proposed therapeutic strategy to avoid the aggregation of Aβ into higher-order structures is to develop molecules that inhibit the early stages of aggregation, i.e., dimerization. Under physiological conditions, the Aβ dimer is highly dynamic and does not attain a single well-defined structure but is rather characterized by an ensemble of conformations. In a recent study, a highly heterogeneous library of conformers of the Aβ dimer was generated by an efficient sampling method with constraints based on ion mobility mass spectrometry data. Here, we make use of the Aβ dimer library to study the interaction with two curcumin degradation products, ferulic aldehyde and vanillin, by molecular dynamics (MD) simulations. Ensemble docking and MD simulations are used to provide atomistic detail of the interactions between the curcumin degradation products and the Aβ dimer. The simulations show that the aromatic residues of Aβ, and in particular 19FF20, interact with ferulic aldehyde and vanillin through π-π stacking. The binding of these small molecules induces significant changes on the 16KLVFF20 region.

Absorption Mode Fourier Transform Ion Mobility Mass Spectrometry Multiplexing Combined with Half-Window Apodization Windows Improves Resolution and Shortens Acquisition Times.

Fourier transform multiplexing enables the coupling of drift tube ion mobility to a wide array of mass spectrometers with improved ion utilization and duty cycles compared to dual-gate signal averaging methods. Traditionally, the data generated by this method is presented in the magnitude mode, but significant improvements in resolution and the signal-to-noise ratio (SNR) are expected if the data can be phase corrected and presented in the absorption mode. A method to simply and reliably determine and correct phase shifts in Fourier transform ion mobility mass spectrometry data using information readily available to any user is presented and evaluated for both small molecule and intact protein analyses with no modification to instrument hardware or experimental procedures. Additionally, the effects of apodization and zero padding are evaluated for both processing methods, and a strategy to use these techniques to reduce acquisition times is presented and evaluated. Resolution is improved by an average factor of 1.6, the SNR is improved by an average factor of 1.2, and acquisition times are reduced by up to 80% through the application of absorption mode processing combined with apodization and zero padding.

Improving computational modeling coupled with ion mobility-mass spectrometry data for efficient drug metabolite structural determination

Ion mobility-mass spectrometry has lately become a widely popular tool for the structural identification of small molecules due to its high efficiency and a low amount requirement for samples. A combination of this technique along with a computational approach has proven to deliver a reliable identification of several compounds that were investigated by comparing experimental and calculated collisional cross sections of structures. Even though a corresponding experimental field has made some valuable developments over the last couple of years, its theoretical counterpart has seen a rather slow improvement. A recent work has introduced a computational protocol for collisional cross-section calculations, but thorough tests have shown that some effort has still to be done to improve its predictive power. The work developed here makes a contribution in that direction by considering possible ways of improving the agreement within different sets of theoretical results as well as between theoretical and experimental values. Special attention has been given to the efficiency of the algorithm along with its large-scale applicability. The importance of internal parameters of the conformational search in the protocol is carefully assessed and new reliable values are suggested.

Predicting the Shapes of Protein Complexes through Collision Cross Section Measurements and Database Searches

In structural biology, collision cross sections (CCSs) from ion mobility mass spectrometry (IM-MS) measurements are routinely compared to computationally or experimentally derived protein structures. Here, we investigate whether CCS data can inform about the shape of a protein in the absence of specific reference structures. Analysis of the proteins in the CCS database shows that protein complexes with low apparent densities are structurally more diverse than those with a high apparent density. Although assigning protein shapes purely on CCS data is not possible, we find that we can distinguish oblate- and prolate-shaped protein complexes by using the CCS, molecular weight, and oligomeric states to mine the Protein Data Bank (PDB) for potentially similar protein structures. Furthermore, comparing the CCS of a ferritin cage to the solution structures in the PDB reveals significant deviations caused by structural collapse in the gas phase. We then apply the strategy to an integral membrane protein by comparing the shapes of a prokaryotic and a eukaryotic sodium/proton antiporter homologue. We conclude that mining the PDB with IM-MS data is a time-effective way to derive low-resolution structural models.

Software Requirements for the Analysis and Interpretation of Native Ion Mobility Mass Spectrometry Data.

The past few years have seen a dramatic increase in applications of native mass and ion mobility spectrometry, especially for the study of proteins and protein complexes. This increase has been catalyzed by the availability of commercial instrumentation capable of carrying out such analyses. As in most fields, however, the software to process the data generated from new instrumentation lags behind. Recently, a number of research groups have started addressing this by developing software, but further improvements are still required in order to realize the full potential of the data sets generated. In this perspective, we describe practical aspects as well as challenges in processing native mass spectrometry (MS) and ion mobility-MS data sets and provide a brief overview of currently available tools. We then set out our vision of future developments that would bring the community together and lead to the development of a common platform to expedite future computational developments, provide standardized processing approaches, and serve as a location for the deposition of data for this emerging field. This perspective has been written by members of the European Cooperation in Science and Technology Action on Native MS and Related Methods for Structural Biology (EU COST Action BM1403) as an introduction to the software tools available in this area. It is intended to serve as an overview for newcomers and to stimulate discussions in the community on further developments in this field, rather than being an in-depth review. Our complementary perspective (http://dx.doi.org/10.1021/acs.analchem.9b05791) focuses on computational approaches used in this field.

Metabolite collision cross section prediction without energy-minimized structures.

Matching experimental ion mobility-mass spectrometry data to computationally-generated collision cross section (CCS) values enables more confident metabolite identifications. Here, we show for the first time that accurately predicting CCS values with simple models for the largest library of metabolite cross sections is indeed possible, achieving a root mean square error of 7.0 Å2 (median error of ∼2%) using linear methods accesible to most researchers. A comparison on the performance of 2D vs. 3D molecular descriptors for the purposes of CCS prediction is also presented for the first time, enabling CCS prediction without a priori knowledge of the metabolite's energy-minimized structure.

Investigating protein patterns in human leukemia cell line experiments: A Bayesian approach for extremely small sample sizes.

Human cancer cell line experiments are valuable for investigating drug sensitivity biomarkers. The number of biomarkers measured in these experiments is typically on the order of several thousand, whereas the number of samples is often limited to one or at most three replicates for each experimental condition. We have developed an innovative Bayesian approach that efficiently identifies clusters of proteins that exhibit similar patterns of expression. Motivated by the availability of ion mobility mass spectrometry data on cell line experiments in myelodysplastic syndrome and acute myeloid leukemia, our methodology can identify proteins that follow biologically meaningful trends of expression. Extensive simulation studies demonstrate good performance of the proposed method even in the presence of relatively small effects and sample sizes.

A strategy for the identification of protein architectures directly from ion mobility mass spectrometry data reveals stabilizing subunit interactions in light harvesting complexes.

Biotechnological applications of protein complexes require detailed information about their structure and composition, which can be challenging to obtain for proteins from natural sources. Prominent examples are the ring‐shaped phycoerythrin (PE) and phycocyanin (PC) complexes isolated from the light‐harvesting antennae of red algae and cyanobacteria. Despite their widespread use as fluorescent probes in biotechnology and medicine, the structures and interactions of their noncrystallizable central subunits are largely unknown. Here, we employ ion mobility mass spectrometry to reveal varying stabilities of the PC and PE complexes and identify their closest architectural homologues among all protein assemblies in the Protein Data Bank (PDB). Our results suggest that the central subunits of PC and PE complexes, although absent from the crystal structures, may be crucial for their stability, and thus of unexpected importance for their biotechnological applications.

Deconvolution of ion mobility mass spectrometry arrival time distributions using a genetic algorithm approach: Application to α1-antitrypsin peptide binding

Ion mobility mass spectrometry (IM-MS) is a fast and sample-efficient method for analysing the gas phase conformation of proteins and protein complexes. Subjecting proteins to increased collision energies prior to ion mobility separation can directly probe their unfolding behaviour. Recent work in the field has utilised this approach to evaluate the effect of small ligand binding upon protein stability, and to screen compounds for drug discovery. Its general applicability for high-throughput screening will, however, depend upon new analytical methods to make the approach scalable. Here we describe a fully automated program, called Benthesikyme, for summarising the ion mobility results from such experiments. The program automatically creates collision induced unfolding (CIU) fingerprints and summary plots that capture the increase in collision cross section and the increase in conformational flexibility of proteins during unfolding. We also describe a program, based on a genetic algorithm, for the deconvolution of arrival time distributions from the CIU data. This multicomponent analysis method was developed to require as little user input as possible. Aside from the IM-MS data, the only input required is an estimate of the number of conformational families to be fitted to the data. In cases where the appropriate number of conformational families is unclear, the automated procedure means it is straightforward to repeat the analysis for several values and optimize the quality of the fit. We have employed our new methodology to study the effects of peptide binding to α1-antitrypsin, an abundant human plasma protein whose misfolding exemplifies a group of conformational diseases termed the serpinopathies. Our analysis shows that interaction with the peptide stabilises the protein and reduces its conformational flexibility. The previously unresolved patterns of unfolding detected by the deconvolution algorithm will allow us to set up a fully automated screen for new ligand molecules with similar properties.

Collision cross section predictions using 2-dimensional molecular descriptors.

Traditional methods for deriving computationally-generated collision cross sections for comparisons with ion mobility-mass spectrometry data require 3-dimensional energy-minimized structures and are often time consuming, preventing high throughput implementation. Here, we introduce a method to predict ion mobility collision cross sections of lipids and peptide analogs important in prebiotic chemistry and other fields. Using less than 100 2-D molecular descriptors this approach resulted in prediction errors of less than 2%.

The Influence of Lipid Bilayer Physicochemical Properties on Gramicidin A Conformer Preferences

The conformational preferences adopted by gramicidin A (GA) dimers inserted into phospholipid bilayers are reported as a function of the bilayer cholesterol content, temperature, and incubation time. Through use of vesicle capture-freeze drying methodology, GA dimers were captured in lipid bilayers and the conformational preferences of the complex were analyzed using ion mobility-mass spectrometry. Perturbations that affect the physicochemical interactions in the lipid bilayer such as cholesterol incorporation, temperature, and incubation time directly alter the conformer preferences of the complex. Regardless of bilayer cholesterol concentration, the antiparallel double helix (ADH) conformation was observed to be most abundant for GA dimers in bilayers composed of lipids with 12 to 22 carbon acyl chains. Incorporation of cholesterol into lipid bilayers yields increased bilayer thickness and rigidity, and an increased abundance of parallel double helix (PDH) and single-stranded head-to-head (SSHH) dimers were observed. Bilayers prepared using 1,2-dilauroyl-sn-glycero-3-phosphocholine, a lipid with 12 carbon acyl chains, yielded a nascent conformer that decreased in abundance as a function of bilayer cholesterol content. High resolution ion mobility-mass spectrometry data revealed two peaks in the ADH region suggesting that ADH populations are composed of two distinct conformers. The conformer preferences of GA dimers from 1,2-distearoyl-sn-glycero-3-phosphocholine bilayers were significantly different for samples incubated at 4°C vs. 60°C; increased cholesterol content yielded more PDH and SSHH at 60°C. The addition of cholesterol as well as incubating samples of 1,2-distearoyl-sn-glycero-3-phosphocholine at 60°C for 24–72 h yielded an increase in PDH and SSHH abundance.

Travelling-wave ion mobility and negative ion fragmentation of high-mannose N-glycans.

The isomeric structure of high-mannose N-glycans can significantly impact biological recognition events. Here, the utility of travelling-wave ion mobility mass spectrometry for isomer separation of high-mannose N-glycans is investigated. Negative ion fragmentation using collision-induced dissociation gave more informative spectra than positive ion spectra with mass-different fragment ions characterizing many of the isomers. Isomer separation by ion mobility in both ionization modes was generally limited, with the arrival time distributions (ATD) often showing little sign of isomers. However, isomers could be partially resolved by plotting extracted fragment ATDs of the diagnostic fragment ions from the negative ion spectra, and the fragmentation spectra of the isomers could be extracted by using ions from limited areas of the ATD peak. In some cases, asymmetric ATDs were observed, but no isomers could be detected by fragmentation. In these cases, it was assumed that conformers or anomers were being separated. Collision cross sections of the isomers in positive and negative fragmentation mode were estimated from travelling-wave ion mobility mass spectrometry data using dextran glycans as calibrant. More complete collision cross section data were achieved in negative ion mode by utilizing the diagnostic fragment ions. Examples of isomer separations are shown for N-glycans released from the well-characterized glycoproteins chicken ovalbumin, porcine thyroglobulin and gp120 from the human immunodeficiency virus. In addition to the cross-sectional data, details of the negative ion collision-induced dissociation spectra of all resolved isomers are discussed.

Biophysical Studies on Interactions and Assembly of Full-size E3 Ubiquitin Ligase: SUPPRESSOR OF CYTOKINE SIGNALING 2 (SOCS2)-ELONGIN BC-CULLIN 5-RING BOX PROTEIN 2 (RBX2)

The multisubunit cullin RING E3 ubiquitin ligases (CRLs) target post-translationally modified substrates for ubiquitination and proteasomal degradation. The suppressors of cytokine signaling (SOCS) proteins play important roles in inflammatory processes, diabetes, and cancer and therefore represent attractive targets for therapeutic intervention. The SOCS proteins, among their other functions, serve as substrate receptors of CRL5 complexes. A member of the CRL family, SOCS2-EloBC-Cul5-Rbx2 (CRL5(SOCS2)), binds phosphorylated growth hormone receptor as its main substrate. Here, we demonstrate that the components of CRL5(SOCS2) can be specifically pulled from K562 human cell lysates using beads decorated with phosphorylated growth hormone receptor peptides. Subsequently, SOCS2-EloBC and full-length Cul5-Rbx2, recombinantly expressed in Escherichia coli and in Sf21 insect cells, respectively, were used to reconstitute neddylated and unneddylated CRL5(SOCS2) complexes in vitro. Finally, diverse biophysical methods were employed to study the assembly and interactions within the complexes. Unlike other E3 ligases, CRL5(SOCS2) was found to exist in a monomeric state as confirmed by size exclusion chromatography with inline multiangle static light scattering and native MS. Affinities of the protein-protein interactions within the multisubunit complex were measured by isothermal titration calorimetry. A structural model for full-size neddylated and unneddylated CRL5(SOCS2) complexes is supported by traveling wave ion mobility mass spectrometry data.

Ion mobility-mass spectrometry of lasso peptides: signature of a rotaxane topology.

Ion mobility mass spectrometry data were collected on a set of five class II lasso peptides and their branched-cyclic topoisomers prepared in denaturing solvent conditions with and without sulfolane as a supercharging agent. Sulfolane was shown not to affect ion mobility results and to allow the formation of highly charged multiply protonated molecules. Drift time values of low charged multiply protonated molecules were found to be similar for the two peptide topologies, indicating the branched-cyclic peptide to be folded in the gas phase into a conformation as compact as the lasso peptide. Conversely, high charge states enabled a discrimination between lasso and branched-cyclic topoisomers, as the former remained compact in the gas phase while the branched-cyclic topoisomer unfolded. Comparison of the ion mobility mass spectrometry data of the lasso and branched-cyclic peptides for all charge states, including the higher charge states obtained with sulfolane, yielded three trends that allowed differentiation of the lasso form from the branched-cyclic topology: low intensity of highly charged protonated molecules, even with the supercharging agent, low change in collision cross sections with increasing charge state of all multiply protonated molecules, and narrow ion mobility peak widths associated with the coexistence of fewer conformations and possible conformational changes.

Collisional and Coulombic Unfolding of Gas‐Phase Proteins: High Correlation to Their Domain Structures in Solution

AbstractThe three‐dimensional structures adopted by proteins are predicated by their many biological functions. Mass spectrometry has played a rapidly expanding role in protein structure discovery, enabling the generation of models for both proteins and their higher‐order assemblies. While important coursed‐grained insights have been generated, relatively few examples exist where mass spectrometry has been successfully applied to the characterization of protein tertiary structure. Here, we demonstrate that gas‐phase unfolding can be used to determine the number of autonomously folded domains within monomeric proteins. Our ion mobility‐mass spectrometry data highlight a strong, positive correlation between the number of protein unfolding transitions observed in the gas phase and the number of known domains within a group of sixteen proteins ranging from 8–78 kDa. This correlation and its potential uses for structural biology is discussed.

Collisional and Coulombic unfolding of gas-phase proteins: high correlation to their domain structures in solution.

The three-dimensional structures adopted by proteins are predicated by their many biological functions. Mass spectrometry has played a rapidly expanding role in protein structure discovery, enabling the generation of models for both proteins and their higher-order assemblies. While important coursed-grained insights have been generated, relatively few examples exist where mass spectrometry has been successfully applied to the characterization of protein tertiary structure. Here, we demonstrate that gas-phase unfolding can be used to determine the number of autonomously folded domains within monomeric proteins. Our ion mobility-mass spectrometry data highlight a strong, positive correlation between the number of protein unfolding transitions observed in the gas phase and the number of known domains within a group of sixteen proteins ranging from 8-78 kDa. This correlation and its potential uses for structural biology is discussed.

Analysis of New Synthetic Drugs by Ion Mobility Time-of-Flight Mass Spectrometry

Characteristic ion mobility mass spectrometry data, reduced mobility, and limits of detection (signal-to-noise ratio = 3) were determined for six synthetic drugs and cocaine by ion mobility time-of-flight mass spectrometry (IM-TOF-MS) with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). The studied synthetic illicit drugs recently appeared on the recreational drug market as designer drugs and were methylone, 4-MEC (4'-methylethcathinone), 3,4-MDPV (3,4-methylenedioxypyrovalerone), JWH-210 [4-ethylnaphthalen-1-yl-(1-pentylindol-3-yl)methanone], JWH-250 [2-(2-methoxyphenyl)-1-(1-pentyl-1H-indol-3-yl)ethanone], and JWH-203 [1-pentyl-3-(2'-chlorophenylacetyl) indole]. Absolute reduced mobilities in nitrogen were 1.35, 1.28, 1.41, 1.30, 1.18, 0.98, 1.09, and 1.07 cm2V(-1)s(-1), for methylone [M-H]+, methylone [M+H]+, 4-MEC [M-H]+, 4-MEC [M+H]+, 3,4-MDPV [M+H]+, JWH-210 [M+H]+, JWH-250 [M+H]+, and JWH-203 [M+H]+, respectively. Selected illicit drugs are easily identified by IM-TOF-MS during a 100s analysis. Relative Limits of detection ranged from 4 to 400 nM are demonstrated for these compounds. Such relative limits of detection correspond to 14 pg to 2 ng absolute limits of detection. Better detection limits are obtained in APCI mode for all the illicit drugs except cocaine. ESI mode was found to be preferable for the IM-TOF-MS detection of cocaine at trace levels. A single sample analysis is completed in an order of magnitude less time than that for conventional liquid chromatography/mass spectrometry approach. The application allows one to consider IM-TOF-MS as a good candidate for a method to determine quickly the recently surfaced designer drugs marketed on the internet as "bath salts," "spice," and "herbal blends".

Gas-phase compaction of helical polymers

To complement the ion mobility mass spectrometry data from the gas-phase experiments on polymers and proteins we have investigated conformations of helical polypeptides by molecular dynamic simulations. Due to strong residue attraction and hydrogen bonding, not mitigated by solvent molecules, a large content of helices, turns and bends was found to persist in long alanine polypeptides (Ala)n even at very high temperatures. As a result, the unsolvated chain dimensions are significantly contracted relative to the random-coil state and the compact structures such as crumpled coils, melted and nematic globules arise at enhanced temperatures. The compactness exponents determined from the power-law plots of the radius of gyration vs lengths suggest that the scaling theory underestimates the polypeptide compaction in the gas phase. At room temperature the polyalanine chains are organized into a variety of helical bundles of antiparallel helical fragments linked by short coils.

Mass Spectrometric Characterization of Protein:Carbohydrate Non Covalent Complexes: What is Fact and What is Fiction

This seminar will present Ion Mobility Mass Spectrometry data showing separation and detection of isomeric glycosaminoglycans (GAG) both with and without protein complexation. Chemokine proteins are involved in inflammation and as such complex with GAG during the activation step of chemotaxis. Comparison of different chemokine conformations suggests that the GAG will complex to different areas of the protein and that sulfation pattern may play a part in complexation. A relatively new modeling program, RAPTOR, is used for determining theoretical cross sections and overall conformation of proteins for which there is no crystal or NMR structure. Both RAPTOR and IM provide valuable information on isomeric separation, identification and conformation of both carbohydrate alone, and with its' protein partner.View Large Image | View Hi-Res Image | Download PowerPoint Slide