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Review
, 1, 293-327

Biomolecule Analysis by Ion Mobility Spectrometry

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Review

Biomolecule Analysis by Ion Mobility Spectrometry

Brian C Bohrer et al. Annu Rev Anal Chem (Palo Alto Calif).

Abstract

Although nonnative protein conformations, including intermediates along the folding pathway and kinetically trapped misfolded species that disfavor the native state, are rarely isolated in the solution phase, they are often stable in the gas phase, where macromolecular ions from electrospray ionization can exist in varying charge states. Differences in the structures of nonnative conformations in the gas phase are often large enough to allow different shapes and charge states to be separated because of differences in their mobilities through a gas. Moreover, gentle collisional activation can be used to induce structural transformations. These new structures often have different mobilities. Thus, there is the possibility of developing a multidimensional separation that takes advantage of structural differences of multiple stable states. This review discusses how nonnative states differ in the gas phase compared with solution and presents an overview of early attempts to utilize and manipulate structures in order to develop ion mobility spectrometry as a rapid and sensitive technique for separating complex mixtures of biomolecules prior to mass spectrometry.

Figures

Figure 1
Figure 1
(a) The relative abundances of three ensembles of structures (native, acid-unfolded, and molten globule) observed by Soret absorption analysis of the acid denaturation of cytochrome c in water. (b) The collision cross sections for the most intense features observed as a function of charge state for protonated cytochrome c in the gas phase. The dotted line denotes the cross section of the native state at 1334 Å2. Figure 1a reprinted from Reference . Copyright American Chemical Society 1993. The value for the dotted line in Figure 1b comes from Reference .
Figure 2
Figure 2
Schematic of the IMS-IMS-IMS-TOF instrument built at Indiana University. Ions are accumulated in the ion funnel F1 and pulsed into the drift tube for experiments by G1. Ions can be mobility selected in two regions (G2, G3); the mobility-selected structures can then be collisionally activated (IA2, IA3). Fragmentation of ions can be performed at IA4, prior to mass analysis in the time-of-flight (TOF) mass analyzer.
Figure 3
Figure 3
Nested td (tf) distribution for the direct infusion of a mixture of ions produced by electrospray of a tryptic peptide mixture from the digestion of dog and pig hemoglobin, bovine and pig albumin, and horse cytochrome c. The solid lines in the two-dimensional plot indicate the positions of the [M + H]+, [M + 2H]2+, and [M + 3H]3+ charge-state families. The vertical line at 18 ms highlights the overlap of different m/z species within the mobility distribution; several of the ions having drift times similar to this value are circled. The numbers and letters label peaks and correspond to the assignments given in table 1 of Reference 25. On the left is a mass spectrum obtained by summing the intensities at a given flight time across all drift time windows. These data were acquired using a drift field of 171.7 V cm−1 with a 300-K helium pressure of 150.3 torr. The data were acquired in ~100 min. Figure adapted from Reference .
Figure 4
Figure 4
Two-dimensional retention time (drift time) contour plots of the high-resolution ion mobility spectrometry–mass spectrometry analysis of a digest of proteins from plasma. (a) The summed intensity of all mass spectral features at each retention time and drift time value. (b) A two-dimensional base-peak plot in which only the most intense mass spectral feature is plotted at each retention time and drift time value. An intensity threshold of four was used to construct both plots. The inset in panel b shows the drift time distribution for species observed at a retention time of 48 min, along with the resolution for several species. Figure reprinted from S.J. Valentine, S.L. Koeniger & D.E. Clemmer, unpublished data.
Figure 5
Figure 5
Drift time distributions of the [M + 7H]7+ of ubiquitin. (a) The total mobility distribution shows that the +7 charge state exists mostly as compact structures (C), some partially folded structures (P), and minimal elongated structures (E). (b) A single peak is observed when a narrow distribution (50 µs) of the compact structure is isolated with mobility selection at 7.8 ms. The inset compares the theoretical to the experimentally measured peak shape. (c) The total mobility distribution for the +7 charge state is reconstructed with 28 mobility-selected distributions acquired every ~0.125 ms. This demonstrates that this distribution arises from many overlapping structures that are stable over the course of the two-dimensional acquisition. Figure reprinted from Reference .
Figure 6
Figure 6
Ion mobility distributions of ubiquitin [M + 7H]7+ ions obtained in IMS-IMS-IMS/MS experiments. The distribution in panel a is the initial distribution consisting of primarily compact conformers. Upon selection of a narrow distribution of compact ions at G2 (100 µs), the distribution in panel b is obtained. Activation of the selected compact ions at IA2 produces the distribution in panel c. A second selection of an intermediate within the partially folded structures performed at G3 (150 µs) is shown in panel d, with the diffusion-limited peak width of the selected ion (gray line). Upon activation of the partially folded structures at IA3, the distribution in panel e is produced, consisting of a broader distribution of partially folded structures and a smaller distribution of elongated states (gray line). The distributions in panels f and g are obtained upon higher-energy activation of the partially folded structures shown in panel d. Figure reprinted from Reference .
Figure 7
Figure 7
(a) An expanded view of an activated selection of human hemoglobin tryptic peptides. The white dotted line denotes the time at which mobility-selected ions with no activation are observed, whereas the dashed yellow lines show the new effective separation space of the second ion mobility spectrometry experiment. Drift distributions for several peptides are shown, along with shifts from original (inactivated) drift times for all ions. (b) Energy versus conformer cross section of the 150 lowest-energy structures post–simulated annealing for two peptides from panel a ([V93 − K99 + 2H]2+ and [T41 − K56 + 3H]3+) as well as cross sections of the energy-minimized structures of the [T41 − K56 + 3H]3+ ion modeled as a helix and linear structure, denoting the range of structures available to each sequence. Cross sections of the selected and activated structures for each sequence are highlighted, along with percent shifts from the mobility-selected structure. Note that the nomenclature used refers to the position of the peptide by providing the location (with respect to the intact protein sequence) and single letter abbreviation of the N- and C-terminal residues, respectively. Figure reprinted from Reference .
Figure 8
Figure 8
(a) Nested plot showing the protonated forms of the dipeptides RA, KV, and LN, which are irresolvable in the drift dimension. (b) The addition of 18-crown-6 ether (18C6) to the electrospray solution creates noncovalent complexes that shift the mobilities of the three peptides from that of the bare species (open white oval) enough that they are now mobility resolved under the same instrumental conditions. Figure reprinted from Reference .
Figure 9
Figure 9
Ion mobility spectrometry–mass spectrometry nested plot of human plasma peptides, electrosprayed from solution containing 18-crown-6 ether (a) with integrated mass spectrum (right column). The white box indicates the region focused on in the other panels of this figure. Mobility-selected complex ions gated through G2 (b) are activated with 160 V at IA2 to dissociate the complex ions into bare peptide ions, which resolve further in mobility (c). Integration of the data between the dashed lines shows that [RHPDYSVVLLLR + 3H]3+ is practically isolated in the drift dimension (c, right column). Parallel dissociation at the end of the drift tube renders mobility-labeled fragments (d). Integration about the dashed lines now provides fragmentation spectrum for [RHPDYSVVLLLR + 3H]3+ (d, right column). Abbreviation: CID, collision-induced dissociation. Figure taken from B.C. Bohrer, S.J. Valentine, S. Naylor & D.E. Clemmer, unpublished manuscript.
Figure 10
Figure 10
(a) Schematic of a circular ion mobility spectrometry time-of-flight instrument. (b) The mobility distribution for mobility-selected bradykinin [M + 2H]2+ ions that have traversed a drift tube containing a single 90° turn. Figure reprinted from S.I. Merenbloom & D.E. Clemmer, unpublished data.

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