Evidence for Many Resolvable Structures within Conformation Types of Electrosprayed Ubiquitin Ions

Abstract

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).

Stepwise evolution of protein native structure with electrospray into the gas phase, 10 −12 to 10 2 s

“…From the 13ϩ ubiquitin ions that apparently have a linear ␣-helical structure, the 11ϩ ions have several bent isomers, the 9ϩ are folded over at one end (stabilized by the opposite dipoles of the overlapping helices), and the 7ϩ, 6ϩ ions have both ends folded over into a three-helix bundle (22) whose compact ion cross-section closely resembles that calculated for the native structure (37)(38)(39)(40). Despite the reduction of total charge, the 7ϩ, 6ϩ conformer (or mixture of similar conformers) has apparently maintained a sufficiently high charge density to retain the IRPD spectral characteristics of extensive hydrogen bonding.…”

“…From IMS (34)(35)(36)(37)(38)(39)(40)45) and ECD (21-23) evidence, H ϩ removal from an ␣-helical peptide or protein ion allows bending at the charge removal site. From the 13ϩ ubiquitin ions that apparently have a linear ␣-helical structure, the 11ϩ ions have several bent isomers, the 9ϩ are folded over at one end (stabilized by the opposite dipoles of the overlapping helices), and the 7ϩ, 6ϩ ions have both ends folded over into a three-helix bundle (22) whose compact ion cross-section closely resembles that calculated for the native structure (37)(38)(39)(40).…”

Effects of ion/ion proton transfer reactions on conformation of gas-phase cytochrome c ions

“…Under these experimental conditions, Cs 7 I 6 ϩ ions or protonated reserpine ions (from ESI of CsI or reserpine in water) yield single mobility peaks ϳ80 s fwhm, roughly half the width of the narrowest protein peaks. Thus, the "single" mobility peaks seen for cytochrome c probably correspond to the juxtaposition of unresolved peaks from ions in several conformations of similar size, as shown by Clemmer et al for ubiquitin [24]. For brevity, we use phrases like "one conformation" or "a single conformation" in the discussion below, with this caveat in mind.…”

“…The development of a 3-D trap-IM-time of flight (TOF) instrument allows time-dependent studies of gas-phase protein ions, including folding, unfolding and structural transitions [20 -23]. A multi-stage IMS-MS instrument [24,25] provides two important new functions. First, a protein ion with a specific structure can be selected by IMS, then activated and separated in the next drift region.…”

Conformational Distribution of Bradykinin [bk + 2 H]2+ Revealed by Cold Ion Spectroscopy Coupled with FAIMS

“…The latter is obtained by "heating" either of the two conformational families prior to the spectroscopic investigation of the gas phase ensemble. Some of the [bk+2 H] 2+ conformations produced by electrospray ionization appear to be kinetically trapped, a situation that may be general for large molecules, where conformational isomerization can be slow [15][16][17][18].…”