Passive transfer of broadly neutralizing HIV antibodies can prevent infection, which suggests that vaccines that elicit such antibodies would be protective. Thus far, however, few broadly neutralizing HIV antibodies that occur naturally have been characterized. To determine whether these antibodies are part of a larger group of related molecules, we cloned 576 new HIV antibodies from four unrelated individuals. All four individuals produced expanded clones of potent broadly neutralizing CD4-binding-site antibodies that mimic binding to CD4. Despite extensive hypermutation, the new antibodies shared a consensus sequence of 68 immunoglobulin H (IgH) chain amino acids and arise independently from two related IgH genes. Comparison of the crystal structure of one of the antibodies to the broadly neutralizing antibody VRC01 revealed conservation of the contacts to the HIV spike.

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".

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 linear octopole trap interface for an ion mobility time-of-flight mass spectrometer has been developed for focusing and accumulating continuous beams of ions produced by electrospray ionization. The interface improves experimental efficiencies by factors of approximately 50-200 compared with an analogous configuration that utilizes a three-dimensional Paul geometry trap (Hoaglund-Hyzer, C. S.; Lee, Y. J.; Counterman, A. E.; Clemmer, D. E. Anal. Chem. 2002, 74, 992-1006). With these improvements, it is possible to record nested drift (flight) time distributions for complex mixtures in fractions of a second. We demonstrate the approach for several well-defined peptide mixtures and an assessment of the detection limits is given. Additionally, we demonstrate the utility of the approach in the field of proteomics by an on-line, three-dimensional nano-LC-ion mobility-TOF separation of tryptic peptides from the Drosophila proteome.

A desorption electrospray ionization (DESI) source has been coupled to an ion mobility time-of-flight mass spectrometer for the analysis of proteins. Analysis of solid-phase horse heart cytochrome c and chicken egg white lysozyme proteins with different DESI solvents and conditions shows similar mass spectra and charge state distributions to those formed when using electrospray to analyze these proteins in solution. The ion mobility data show evidence for compact ion structures [when the surface is exposed to a spray that favors retention of "nativelike" structures (50:50 water:methanol)] or elongated structures [when the surface is exposed to a spray that favors "denatured" structures (49:49:2 water:methanol:acetic acid)]. The results suggest that the DESI experiment is somewhat gentler than ESI and under appropriate conditions, it is possible to preserve structural information throughout the DESI process. Mechanisms that are consistent with these results are discussed.