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.

The development of a new ion mobility/mass spectrometry instrument that incorporates a multifield drift tube/ion funnel design is described. In this instrument, individual components from a mixture of ions can be resolved and selected on the basis of mobility differences prior to collisional activation inside the drift tube. The fragment ions that are produced can be dispersed again in a second ion mobility spectrometry (IMS) region prior to additional collisional activation and MS analysis. The result is an IMS-IMS analogue of MS-MS. Here, we describe the preliminary instrumental design and experimental approach. We illustrate the approach by examining the highly characterized bradykinin and ubiquitin systems. Mobility-resolved fragment ions of bradykinin show that b-type ions are readily discernible fragments, because they exist as two easily resolvable structural types. Current limitations and future directions are briefly discussed.

A new two-dimensional ion mobility spectrometry approach combined with mass spectrometry has been used to examine ubiquitin ions in the gas phase. In this approach ions are separated in an initial drift tube into conformation types (defined by their collision cross sections) and then a gate is used to introduce a narrow distribution of mobility-separated ions into a second drift tube for subsequent separation. The results show that upon selection a narrow peak shape is retained through the second drift tube. This requires that at 300 K the selected distribution does not interconvert substantially within the broader range of structures associated with the conformation type within the approximately 10-20 ms time scale of these experiments. For the [M + 7H]7+ ion, it appears that many ( approximately 5-10) narrow selections can be made across each of the compact, partially-folded, and elongated conformer types, defined previously (Int. J. Mass Spectrom. 1999, 187, 37-47).

Multidimensional ion mobility spectrometry (IMS-IMS and IMS-IMS-IMS) techniques have been combined with mass spectrometry (MS) and investigated as a means of generating and separating peptide and protein fragment ions. When fragments are generated inside a drift tube and then dispersed by IMS prior to MS analysis, it is possible to observe many features that are not apparent from MS analysis alone. The approach is demonstrated by examining fragmentation patterns arising from electrospray ion distributions of insulin chain B and ubiquitin. The multidimensional IMS approach makes it possible to select individual components for collisional activation and to disperse fragments based on differences in mobility prior to MS analysis. Such an approach makes it possible to observe many features not apparent by MS analysis alone.

Effective temperatures of ions during traveling wave ion mobility spectrometry (TWIMS) analysis were measured using singly protonated leucine enkephalin dimer as a chemical thermometer by monitoring dissociation of the dimer into monomer, as well as the subsequent dissociation of monomer into a-, b-, and y-ions, as a function of instrumental parameters. At fixed helium cell and TWIMS cell gas flow rates, the extent of dissociation does not vary significantly with either the wave velocity or wave height, except at low (<500 m/s) wave velocities that are not commonly used. Increasing the flow rate of nitrogen gas into the TWIMS cell and decreasing the flow rate of helium gas into the helium cell resulted in greater dissociation. However, the mobility distributions of the fragment ions formed by dissociation of the dimer upon injection into the TWIMS cell are nearly indistinguishable from those of fragment ions formed in the collision cell prior to TWIMS analysis for all TWIMS experiments. These results indicate that heating and dissociation occur when ions are injected into the TWIMS cell, and that the effective temperature subsequently decreases to a point at which no further dissociation is observed during the TWIMS analysis. An upper limit to the effective ion temperature of 449 K during TWIMS analysis is obtained at a helium flow rate of 180 mL/min, TWIMS flow rate of 80 mL/min and traveling wave height of 40 V, which is well below previously reported values. Effects of ion heating in TWIMS on gas-phase protein conformation are presented.

A novel ion mobility spectrometry instrument incorporating a cyclotron geometry drift tube is presented. The drift tube consists of eight regions, four curved drift tubes and four ion funnels. Packets of ions are propagated around the drift tube by changing the drift field at a frequency that is resonant with the ion's drift time through each region. The approach trims each packet of ions as it leaves and enters each new region. An electrostatic gate allows ions to be kept in the drift tube for numerous cycles, increasing the ability to resolve specified ions. We demonstrate the approach by isolating the [M+2H] 2+ or [M+3H] 3+ charge state of substance P as well as individual trisaccharide isomers from a mixture of melezitose and raffinose. Resolving powers in excess of 300 are obtainable with this approach.

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Multidimensional ion mobility spectrometry techniques (IMS-IMS and IMS-IMS-IMS) combined with mass spectrometry are used to study structural transitions of ubiquitin ions in the gas phase. It is possible to select and activate narrow distributions of compact and partially folded conformation types and examine new distributions of structures that are formed. Different compact conformations unfold, producing a range of new partially folded states and three resolvable peaks associated with elongated conformers. Under gentle activation conditions, the final populations of the three elongated forms depend on the initial structures of the selected ions. This requires that some memory of the compact state (most likely secondary structure) is preserved along the unfolding pathway. Activation of selected, partially folded intermediates (formed from specific compact states) leads to elongated state populations that are consistent with the initial selected compact form-evidence that intermediates not only retain elements of initial structure but also are capable of transmitting structure to final states.