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.

Electrospray ionization, combined with two-dimensional ion mobility spectrometry and mass spectrometry, is used to produce, select, and activate distributions of elongated ions, [M ϩ 11H] 11ϩ to [M ϩ 13H] 13ϩ , of ubiquitin. The analysis makes it possible to examine state-to-state transitions for structural types, and transition diagrams associated with the efficiencies of structural changes are presented. The ϩ11 and ϩ12 charge states can form four resolvable states while only one state is formed for [M ϩ 13H] 13ϩ . Some conformations, which appear to belong to the same family based on mobility analysis of different charge states, undergo similar transitions, others do not. Activation of ions that exist in low-abundance conformations, having mobilities that fall in between sharp peaks associated with higher abundances species, shows that the low-abundance forms undergo efficient (ϳ90 to 100%) conversion into states associated with well-defined peaks. This efficiency is significantly higher than the ϳ10 to 60% efficiency of transitions of structures associated with well-defined peaks. The formation of sharp features from a range of low-intensity species with different cross sections indicates that large regions of conformation space must be unfavorable or inaccessible in the gas phase. These results are compared with several previous IMS measurements of this system as well as information about gas-phase structure provided by other techniques. Studies of solvent-free proteins and peptides are important because of both fundamental and practical considerations. In the absence of solvation shells (or with minimal solvent), it is possible to extract kinetic and thermodynamic benchmarks about the formation of specific types of folds and tease out the influence of solvent-molecule and intramolecular factors in establishing conformation [2][3][4][5][6][7][8][9][10][11]. From a practical standpoint, a basic understanding of gas-phase macromolecular conformation is important because it is largely independent of the ion mass-to-charge (m/z) ratio. Therefore, assessing the conformations of gas-phase ions provides a means of complementing structural information obtained from MS measurements [12][13][14][15][16][17][18][19][20].A number of groups have worked to combine ion mobility spectrometry (IMS) and MS with the aim of using differences in ion mobility to separate components of a mixture that would not be resolved by MS alone [21, 22]. The mobility of a macromolecular ion through a buffer gas depends on its charge and shape (average collision cross section with the buffer gas, ⍀). Recently, we have ex- [23][24][25][26]. One reason for developing these instruments is that it is possible to separate a distribution of initial conformations produced by an ion source, select a structural ion type (based on its mobility), and then activate the selected ions to induce transitions before characterizing the ions by separation through another drift region. By changing the structures of the ions, it is possible to resolve fea...