Structural transitions of the +6 to +8 charge states of ubiquitin produced by electrospray ionization have been studied in the gas phase by a new ion trap/ion mobility-mass spectrometry technique. The approach allows transitions to be examined in detail over ∼10 ms to 30 s trapping times. This time regime is intermediate between the ∼1 to 5 ms time scales of previous mobility measurements [J. Am. Soc. Mass Spectrom. 1997, 8, 954] and minute to hour time scale measurements associated with trapping experiments done in a Fourier transform mass spectrometer [Int. J. Mass Spectrom. 1999, 185/186/187, 565]. The results show that over the entire time range, the +6 charge state is dominated by compact structures (with cross sections that are near the value expected for folded states in solution). The +7 state shows evidence for at least two types of initial compact structures. One state (∼65% of the population) rapidly unfolds to partially folded and elongated conformers after ∼30 to 40 ms. The remaining 35% of ions also unfolds at a much slower rate. The +8 charge state appears to be formed initially in a range of partially folded states. These states rapidly unfold into elongated structures that persist to the longest trapping times that are employed. These results are compared with the longer time scale measurements, and attempts are made to correlate the features observed in the different experiments. † Part of the special issue "Jack Beauchamp Festschrift".

A new technique for studying the time dependence of conformational changes of gas-phase protein ions is described. In this approach, a short pulse of electrosprayed protein ions is introduced into an ion trap and stored. After a defined time period, the distribution of ions is ejected from the trap into an ion mobility/time-of-flight mass spectrometer. Combined measurements of mobilities and flight times in the mass spectrometer provide information about the abundances of different conformer types and charge-state distributions. By varying the storage time in the trap, it is possible to monitor changes in ion conformation that occur over extended time periods (approximately 10-200 ms). The method is demonstrated by examining changes in cytochrome c ion conformations for the +7 to +10 charge states.

The folding pathways of gas-phase cytochrome c ions produced by electrospray ionization have been studied by an ion trapping/ion mobility technique that allows conformations to be examined over extended timescales (10 ms to 10 s). The results show that the ϩ9 charge state emerges from solution as a compact structure and then rapidly unfolds into several substantially more open structures, a transition that requires 30-60 ms; over substantially longer timescales (250 ms to 10 s) elongated states appear to refold into an array of folded structures. The new folded states are less compact than those that are apparent during the initial unfolding. Apparently, unfolding to highly open conformations is a key step that must occur before ϩ9 ions can sample more compact states that are stable at longer times. . Recently, structural studies of ions in the gas phase have attracted significant attention as a means of studying intrinsic interactions of biomolecules in the absence of solvent to further understand fundamental aspects of protein folding. Examination of gas-phase structure/ conformations of protein ions has primarily been studied by ion mobility [4, 5] and gas-phase hydrogendeuterium exchange [6-10] methods, with some recent studies [11,12] using the "gentle" dissociation process of electron capture dissociation (ECD) [13]. In addition, unfolding and folding transitions of gas-phase ions have been studied by exciting the ions via laser irradiation [8,11,12] or energetic collisions [5,8,9] followed by analysis of structure/conformation by the aforementioned methods. The advantages of studying gas-phase ions is that one is able to study not only "naked" ions' intrinsic physical and chemical properties, but also the stepwise solvation of these ions [14 -16].We have recently developed a new method for studying the time-dependent behavior of gas-phase protein ions. In this approach we store ions in a Paul geometry ion trap for variable amounts of time and follow the structural transitions by analyzing the conformations of electrosprayed ions by a combined ion mobility/time-of-flight analysis [17,18]. In this study we show evidence for structural transitions of the ϩ9

A cylindrical geometry ion trap is used to record mass spectra in the mass-selective instability mode. The geometry of the cylindrical ion trap has been optimized to maximize the quadrupole field component relative to the higher-order field content through field calculations using the Poisson/Superfish code and through experimental variation of the electrode structure. The results correspond well with predictions of the calculations. The trap has been used to record mass spectra with better than unit mass resolution, high sensitivity, and a mass/charge range of ∼600 Th. Multistage (MS(3)) experiments have been performed, and the Mathieu stability region has been experimentally mapped. The performance of this device compares satisfactorily with that of the hyperbolic ion trap.

A cylindrical quadrupole ion trap (r(0) = 2.5 mm, z(0) = 2.88 mm, ∼(1)/(64) of the volume of commercial hyperbolic ion traps) has been constructed, its geometry optimized, and its performance examined in the mass-selective instability scan mode. Spectra of ionized perfluorotributylamine and o-dichlorobenzene show a resolution (m/Δm, 50% valley definition) of ∼100. The instrument has been coupled to a membrane introduction system to test its applicability for on-line reaction monitoring and to determine detection limits. Simulations using the ion trap simulation program are used to explore the effects of geometry on performance and to validate the experimental results.