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 (
≈ 3.0 to 1.2 cm
/(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.,
≈ 3.0 to 1.67 cm
/(V*s)) was achieved, with transmission efficiency rolling off for the lower mobility ions (e.g.,
≈ 1.2 cm
/(V*s)). Resolving powers of up to ∼560 were achieved using all four ion levels to separate reverse peptides (SDGRG
and GRGDS
). 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.
Selected Overtone Mobility Spectrometry Ewing, Michael A; Conant, Christopher R. P; Zucker, Steven M ...
Analytical chemistry (Washington),
05/2015, Letnik:
87, Številka:
10
Journal Article
Recenzirano
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.
The unanticipated discovery of recent ultra-high-resolution ion mobility spectrometry (IMS) measurements revealing that isotopomerscompounds that differ only in the isotopic substitution sitescan ...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.
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.
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.
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+2H2++H+ → BK+3H3+, 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+3H3+ 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: ΔG ‡ = 94.8 ± 0.2 kJ·mol–1, ΔH ‡ = 79.8 ± 0.2 kJ·mol–1, and ΔS ‡ = −50.4 ± 1.7 J·mol–1·K–1 for the trans → cis protonation event; and, ΔG ‡ = 94.1 ± 9.2 kJ·mol–1, ΔH ‡ = 107.3 ± 9.2 kJ·mol–1, and ΔS ‡ = 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.
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.
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 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.
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–Gly n –Lys n+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.
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