AutoCCS: automated collision cross-section calculation software for ion mobility spectrometry-mass spectrometry.

MOTIVATION: Ion mobility spectrometry (IMS) separations are increasingly used in conjunction with mass spectrometry (MS) for separation and characterization of ionized molecular species. Information obtained from IMS measurements includes the ion's collision cross section (CCS), which reflects its size and structure and constitutes a descriptor for distinguishing similar species in mixtures that cannot be separated using conventional approaches. Incorporating CCS into MS-based workflows can improve the specificity and confidence of molecular identification. At present, there is no automated, open-source pipeline for determining CCS of analyte ions in both targeted and untargeted fashion, and intensive user-assisted processing with vendor software and manual evaluation is often required. RESULTS: We present AutoCCS, an open-source software to rapidly determine CCS values from IMS-MS measurements. We conducted various IMS experiments in different formats to demonstrate the flexibility of AutoCCS for automated CCS calculation: (i) stepped-field methods for drift tube-based IMS (DTIMS), (ii) single-field methods for DTIMS (supporting two calibration methods: a standard and a new enhanced method) and (iii) linear calibration for Bruker timsTOF and non-linear calibration methods for traveling wave based-IMS in Waters Synapt and Structures for Lossless Ion Manipulations. We demonstrated that AutoCCS offers an accurate and reproducible determination of CCS for both standard and unknown analyte ions in various IMS-MS platforms, IMS-field methods, ionization modes and collision gases, without requiring manual processing. AVAILABILITY AND IMPLEMENTATION: https://github.com/PNNL-Comp-Mass-Spec/AutoCCS. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. Demo datasets are publicly available at MassIVE (Dataset ID: MSV000085979).

Measurement and Theory of Gas-Phase Ion Mobility Shifts Resulting from Isotopomer Mass Distribution Changes.

The unanticipated discovery of recent ultra-high-resolution ion mobility spectrometry (IMS) measurements revealing that isotopomers─compounds that differ only in the isotopic substitution sites─can be separated has raised questions as to the physical basis for their separation. A study comparing IMS separations for two isotopomer sets in conjunction with theory and simulations accounting for ion rotational effects provides the first-ever prediction of rotation-mediated shifts. The simulations produce observable mobility shifts due to differences in gas-ion collision frequency and translational-to-rotational energy transfer. These differences can be attributed to distinct changes in the moment of inertia and center of mass between isotopomers. The simulations are in broad agreement with the observed experiments and consistent with relative mobility differences between isotopomers. These results provide a basis for refining IMS theory and a new foundation to obtain additional structural insights through IMS.

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.

Ultra-High-Resolution Ion Mobility Separations Over Extended Path Lengths and Mobility Ranges Achieved using a Multilevel Structures for Lossless Ion Manipulations Module.

Over the past few years, structures for lossless ion manipulations (SLIM) have used traveling waves (TWs) to move ions over long serpentine paths that can be further lengthened by routing the ions through multiple passages of the same path. Such SLIM "multipass" separations provide unprecedentedly high ion mobility resolving powers but are ultimately limited in their ion mobility range because of the range of mobilities spanned in a single pass; that is, higher mobility ions ultimately "overtake" and "lap" lower mobility ions that have experienced fewer passes, convoluting their arrival time distribution at the detector. To achieve ultrahigh resolution separations over broader mobility ranges, we have developed a new multilevel SLIM possessing multiple stacked serpentine paths. Ions are transferred between SLIM levels through apertures (or ion escalators) in the SLIM surfaces. The initial multilevel SLIM module incorporates four levels and three interlevel ion escalator passages, providing a total path length of 43.2 m. Using the full path length and helium buffer gas, high resolution separations were achieved for Agilent tuning mixture phosphazene ions over a broad mobility range (K0 ≈ 3.0 to 1.2 cm2/(V*s)). High sensitivity was achieved using "in-SLIM" ion accumulation over an extended trapping region of the first SLIM level. High transmission efficiency of ions over a broad mobility range (e.g., K0 ≈ 3.0 to 1.67 cm2/(V*s)) was achieved, with transmission efficiency rolling off for the lower mobility ions (e.g., K0 ≈ 1.2 cm2/(V*s)). Resolving powers of up to ∼560 were achieved using all four ion levels to separate reverse peptides (SDGRG1+ and GRGDS1+). A complex mixture of phosphopeptides showed similar coverage could be achieved using one or all four SLIM levels, and doubly charged phosphosite isomers not significantly separated using one SLIM level were well resolved when four levels were used. The new multilevel SLIM technology thus enables wider mobility range ultrahigh-resolution ion mobility separations and expands on the ability of SLIM to obtain improved separations of complex mixtures with high sensitivity.

Monitoring the stabilities of a mixture of peptides by mass-spectrometry-based techniques.

Biomolecular degradation plays a key role in proteostasis. Typically, proteolytic enzymes degrade proteins into smaller peptides by breaking amino acid bonds between specific residues. Cleavage around proline residues is often missed and requires highly specific enzymes for peptide processing due to the cyclic proline side-chain. However, degradation can occur spontaneously (i.e. in the absence of enzymes). In this study, the influence of the first residue on the stability of a series of penultimate proline containing peptides, with the sequence Xaa-Pro-Gly-Gly (where Xaa is any amino acid), is investigated with mass spectrometry techniques. Peptides were incubated as mixtures at various solution temperatures (70℃ to 90℃) and were periodically sampled over the duration of the experiment. At elevated temperatures, we observe dissociation after the Xaa-Pro motif for all sequences, but at different rates. Transition state thermochemistry was obtained by studying the temperature-dependent kinetics and although all peptides show relatively small differences in the transition state free energies (∼95 kJ/mol), there is significant variability in the transition state entropy and enthalpy. This demonstrates that the side-chain of the first amino acid has a significant influence on the stability of the Xaa-Pro sequence. From these data, we demonstrate the ability to simultaneously measure the dissociation kinetics and relative transition state thermochemistries for a mixture of peptides, which vary only in the identity of the N-terminal amino acid.

Conformationally Regulated Peptide Bond Cleavage in Bradykinin.

Ion mobility and mass spectrometry techniques are used to investigate the stabilities of different conformations of bradykinin (BK, Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9). At elevated solution temperatures, we observe a slow protonation reaction, i.e., [BK+2H]2++H+ → [BK+3H]3+, that is regulated by trans → cis isomerization of Arg1-Pro2, resulting in the Arg1- cis-Pro2- cis-Pro3-Gly4-Phe5-Ser6- cis-Pro7-Phe8-Arg9 (all- cis) configuration. Once formed, the all- cis [BK+3H]3+ spontaneously cleaves the bond between Pro2-Pro3 with perfect specificity, a bond that is biologically resistant to cleavage by any human enzyme. Temperature-dependent kinetics studies reveal details about the intrinsic peptide processing mechanism. We propose that nonenzymatic cleavage at Pro2-Pro3 occurs through multiple intermediates and is regulated by trans → cis isomerization of Arg1-Pro2. From this mechanism, we can extract transition state thermochemistry: Δ G‡ = 94.8 ± 0.2 kJ·mol-1, Δ H‡ = 79.8 ± 0.2 kJ·mol-1, and Δ S‡ = -50.4 ± 1.7 J·mol-1·K-1 for the trans → cis protonation event; and, Δ G‡ = 94.1 ± 9.2 kJ·mol-1, Δ H‡ = 107.3 ± 9.2 kJ·mol-1, and Δ S‡ = 44.4 ± 5.1 J·mol-1·K-1 for bond cleavage. Biological resistance to the most favored intrinsic processing pathway prevents formation of Pro3-Gly4-Phe5-Ser6- cis-Pro7-Phe8-Arg9 that is approximately an order of magnitude more antigenic than BK.

Selected overtone mobility spectrometry.

A new means of acquiring overtone mobility spectrometry (OMS) data sets that allows distributions of ions for a prescribed overtone number is described. In this approach, the drift fields applied to specific OMS drift regions are varied to make it possible to select different ions from a specific overtone that is resonant over a range of applied frequencies. This is accomplished by applying different fields for fixed ratios of time while scanning the applied frequency. The ability to eliminate peaks from all but a single overtone region overcomes a significant limitation associated with OMS analysis of unknowns, especially in mixtures. Specifically, a priori knowledge via selection of the overtone used to separate ions makes it possible to directly determine ion mobilities for unknown species and collision cross sections (assuming that the ion charge state is known). We refer to this selection method of operation as selected overtone mobility spectrometry (SOMS). A simple theoretical description of the SOMS approach is provided. Simulations are carried out and discussed in order to illustrate the advantages and disadvantages of SOMS compared with traditional OMS. Finally, the SOMS method (and its distinction from OMS) is demonstrated experimentally by examining a mixture of peptides generated by enzymatic digestion of the equine cytochrome c with trypsin.

Implementation of Ion Mobility Spectrometry-Based Separations in Structures for Lossless Ion Manipulations (SLIM).

Structures for Lossless Ion Manipulations (SLIM) is a powerful variant of traveling wave ion mobility spectrometry (TW-IMS) that uses a serpentine pattern of microelectrodes deposited onto printed circuit boards to achieve ultralong ion path lengths (13.5 m). Ions are propelled through SLIM platforms via arrays of TW electrodes while RF and DC electrodes provide radial confinement, establishing near lossless transmission. The recent ability to cycle ions multiple times through a SLIM has allowed ion path lengths to exceed 1000 m, providing unprecedented separation power and the ability to observe ion structural conformations unobtainable with other IMS technologies. The combination of high separation power, high signal intensity, and the ability to couple with mass spectrometry places SLIM in the unique position of being able to address longstanding proteomics and metabolomics challenges by allowing the characterization of isomeric mixtures containing low abundance analytes.

Diketopiperazine Formation from FPGnK (n = 1-9) Peptides: Rates of Structural Rearrangements and Mechanisms.

Peptides with penultimate proline residues undergo trans → cis isomerization of the Phe1-Pro2 peptide bond followed by spontaneous bond cleavage at the Pro2-Xxx3 bond (where Xxx is another amino acid residue), leading to cleavage of the Pro2-Xxx3 bond and formation of a diketopiperazine (DKP). In this paper, ion mobility spectrometry and mass spectrometry techniques were used to study the dissociation kinetics of nine peptides [Phe1-Pro2-Glyn-Lysn+3 (n = 1-9)] in ethanol. Shorter (n = 1-3) peptides are found to be more stable than longer (n = 4-9) peptides. Alanine substitution studies indicate that, when experiments are initiated, the Phe1-Pro2 bond of the n = 9 peptide exists exclusively in the cis configuration, while the n = 1-8 peptides appear to exist initially with both cis- and trans-Phe1-Pro2 configured bonds. Molecular dynamics simulations indicate that intramolecular hydrogen bonding interactions stabilize conformations of shorter peptides, thus inhibiting DKP formation. Similar stabilizing interactions appear less frequently in longer peptides. In addition, in smaller peptides, the N-terminal amino group is more likely to be charged compared to the same group in longer peptides, which would inhibit the dissociation through the DKP formation mechanism. Analysis of temperature-dependent kinetics measurements provides insight about the mechanism of bond cleavage. The analysis gives the following transition state thermochemistry: ΔG⧧ values range from 94.6 ± 0.9 to 101.5 ± 1.9 kJ·mol-1, values of ΔH⧧ range from 89.1 ± 0.9 to 116.7 ± 1.5 kJ·mol-1, and ΔS⧧ values range from -25.4 ± 2.6 to 50.8 ± 4.2 J·mol-1·K-1. Proposed mechanisms and thermochemistry are discussed.

Evaluation of Waveform Profiles for Traveling Wave Ion Mobility Separations in Structures for Lossless Ion Manipulations.

Structures for lossless ion manipulations (SLIM) have recently enabled a powerful implementation of traveling wave ion mobility spectrometry (TWIMS) for ultrahigh resolution separations; however, experimental parameters have not been optimized, and potential significant gains may be feasible. Most TWIMS separations have utilized square-shaped waveforms applied by time-dependent voltage stepping across repeating sets of electrodes, but alternative waveforms may provide further improvements to resolution. Here, we characterize five waveforms (including square and sine) in terms of their transmission efficiency, IMS resolution, and resolving power, and explore the effects of TW amplitude and speed on the performance of each. We found, consistent with previous work, separations were generally improved with higher TW amplitudes, moderately improved by lower speeds (limited by ion "surfing" with the waves), and found decreases in signal intensity at the extremes of operating conditions. The triangle and asymmetric "ramp forward" shaped profiles were found to provide modestly greater resolution and resolving power, an observation we tentatively attribute to their relatively uniform fields and minimal low-field regions.

Dynamic Time-Warping Correction for Shifts in Ultrahigh Resolving Power Ion Mobility Spectrometry and Structures for Lossless Ion Manipulations.

Detection of arrival time shifts between ion mobility spectrometry (IMS) separations can limit achievable resolving power (Rp), particularly when multiple separations are summed or averaged, as commonly practiced in IMS. Such variations can be apparent in higher Rp measurements and are particularly evident in long path length traveling wave structures for lossless ion manipulations (SLIM) IMS due to their typically much longer separation times. Here, we explore data processing approaches employing single value alignment (SVA) and nonlinear dynamic time warping (DTW) to correct for variations between IMS separations, such as due to pressure fluctuations, to enable more effective spectrum summation for improving Rp and detection of low-intensity species. For multipass SLIM IMS separations, where narrow mobility range measurements have arrival times that can extend to several seconds, the SVA approach effectively corrected for such variations and significantly improved Rp for summed separations. However, SVA was much less effective for broad mobility range separations, such as obtained with multilevel SLIM IMS. Changes in ions' arrival times were observed to be correlated with small pressure changes, with approximately 0.6% relative arrival time shifts being common, sufficient to result in a loss of Rp for summed separations. Comparison of the approaches showed that DTW alignment performed similarly to SVA when used over a narrow mobility range but was significantly better (providing narrower peaks and higher signal intensities) for wide mobility range data. We found that the DTW approach increased Rp by as much as 115% for measurements in which 50 IMS separations over 2 s were summed. We conclude that DTW is superior to SVA for ultra-high-resolution broad mobility range SLIM IMS separations and leads to a large improvement in effective Rp, correcting for ion arrival time shifts regardless of the cause, as well as improving the detectability of low-abundance species. Our tool is publicly available for use with universal ion mobility format (.UIMF) and text (.txt) files.

Substance P in the Gas Phase: Conformational Changes and Dissociations Induced by Collisional Activation in a Drift Tube.

The work presented below is related to our companion paper in this issue, entitled: Substance P in solution: trans-to-cis configurational changes of penultimate prolines initiate non-enzymatic peptide bond cleavages. Two-dimensional ion mobility spectrometry (IMS-IMS) and mass spectrometry techniques are used to investigate structural transitions for [M+3H]3+ ions of substance P (subP) upon collisional activation (CA) in the gas phase. In this approach, different conformations of ions having a specified mobility are selected after an initial IMS separation, collisionally activated to produce new conformers, and these product structures are separated again using a second IMS region. In this way, it is possible to follow folding and unfolding transitions of different conformations. The analysis shows evidence for five conformations. Unlike other systems, every transition is irreversible. Studies as a function of activation voltage are used to discern pathways of structural changes prior to reaching the energy required for dissociation. Thresholds associated with the onsets of transitions are calibrated to obtain estimates of the energetic barriers between different structures and semi-quantitative potential energy diagrams are presented. Overall, barriers associated with structural transitions of [subP+3H]3+ in the absence of solvent are on the order of ~ 40 kJ mol-1, substantially lower than the ~ 90 kJ mol-1 required for some similar structural transitions in solutions of ethanol. Comparisons of the transition energies in the gas phase with thermochemistry for similar transitions in solution provide clues about why reverse transitions are prohibited. Graphical Abstract.

Substance P in Solution: Trans-to-Cis Configurational Changes of Penultimate Prolines Initiate Non-enzymatic Peptide Bond Cleavages.

We report ion mobility spectrometry and mass spectrometry studies of the non-enzymatic step-by-step degradation of substance P (subP), an 11-residue neuropeptide, with the sequence Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7-Phe8-Gly9-Leu10-Met11-NH2, in ethanol. At elevated solution temperatures (55 to 75 °C), several reactions are observed, including a protonation event, i.e., [subP+2H]2+ + H+ → [subP+3H]3+, that appears to be regulated by a configurational change and two sequential bond cleavages (the Pro2-Lys3 peptide bond is cleaved to form the smaller nonapeptide Lys3-Met11-NH2 [subP(3-11)], and subsequently, subP(3-11) is cleaved at the Pro4-Gln5 peptide bond to yield the heptapeptide Gln5-Met11-NH2 [subP(5-11)]). Each of the product peptides [subP(3-11) and subP(5-11)] is accompanied by a complementary diketopiperazine (DKP): cyclo-Arg1-Pro2 (cRP) for the first cleavage, and cyclo-Lys3-Pro4 (cKP) for the second. Insight about the mechanism of degradation is obtained by comparing kinetics calculations of trial model mechanisms with experimental data. The best model of our experimental data indicates that the initial cleavage of subP is regulated by a conformational change, likely a trans→cis isomerization of the Arg1-Pro2 peptide bond. The subP(3-11) product has a long lifetime (t1/2 ~ 30 h at 55 °C) and appears to transition through several structural intermediates prior to dissociation, suggesting that subP(3-11) is initially formed with a Lys3-trans-Pro4 peptide bond configuration and that slow trans→cis isomerization regulates the second bond cleavage event as well. From these data and our model mechanisms, we obtain transition state thermochemistry ranging from ΔH‡ = 41 to 85 kJ mol-1 and ΔS‡ = - 43 to - 157 J mol-1 K-1 for each step in the reaction. Graphical Abstract.

Melting proteins confined in nanodroplets with 10.6 µm light provides clues about early steps of denaturation.

Ubiquitin confined within nanodroplets was irradiated with a variable-power CO2 laser. Mass spectrometry analysis shows evidence for a protein "melting"-like transition within droplets prior to solvent evaporation and ion formation. Ion mobility spectrometry reveals that structures associated with early steps of denaturation are trapped because of short droplet lifetimes.