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

Abstract

Positive ions from cytochrome c are studied in a 3-D ion trap/ion mobility (IM)/quadrupoletime-of-flight (TOF) instrument with three independent ion sources. The IM separation allows measurement of the cross section of the ions. Ion/ion reactions in the 3-D ion trap that remove protons cause the cytochrome c ions to refold gently without other degradation of protein structure, i.e., fragmentation or loss of heme group or metal ion. The conformation(s) of the product ions generated by ion/ion reactions in a given charge state are similar regardless of whether the cytochrome c ions are originally in ϩ8 or ϩ9 charge states. In the lower charge states (ϩ1 to ϩ5) cytochrome c ions made by the ion/ion reaction yield a single IM peak with cross section of ϳ1110 to 1180 Å 2 , even if the original ϩ8 ion started with multiple conformations. The conformation expands slightly when the charge state is reduced from ϩ5 to ϩ1. For product ions in the ϩ6 to ϩ8 charge states, ions created from higher charge states (ϩ9 to ϩ16) by ion/ion reaction produce more compact conformation(s) in somewhat higher abundances compared with those produced directly by the electrospray ionization ( [5][6][7][8][9], and native electron capture dissociation (NECD) [10,11]. Of these methods, IM provides a direct way to examine the gas-phase conformation of the ions by probing the average cross-section of the protein ions via collisions with buffer gas [3,4]. Early IM research on protein folding and unfolding was done with an IMquadrupole instrument [1]. To study ions in lower charge states than those made directly from the electrospray ionization (ESI) source, a basic collision gas (e.g., acetophenone or 7-methyl-1,3,5-triazabicyclo 4,4,0] dec-5-ene, MTBD) [12] was introduced into the source region. The neutral gas extracted protons from the protein ion and created lower charge state ions through proton transfer reactions in the source. In these studies, the reactions took place only in the atmospheric pressure ion source interface region. Thus, control and variation of the reaction time and extent of reaction were difficult, and only certain reagent species were available.Gas-phase ion/ion reactions provide another dimension for gas-phase bioanalysis by MS. To date, these reactions have been mainly used to simplify complex MS/MS spectra [13,14] or generate fragments for structural assignment [15,16] by methods like electrontransfer dissociation (ETD) [17][18][19].Recent instrumentation improvements have greatly extended the type of structural information and number of possible experiments available in this area. 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. Second, "state-to-state" structural

Ion Mobility Measurements of Nondenatured 12–150 kDa Proteins and Protein Multimers by Tandem Differential Mobility Analysis–Mass Spectrometry (DMA-MS)

“…With the DMA-MS, we are able to clearly determine protein ion mobilities (in dry air) and masses simultaneously, from which we infer their spherical-equivalent diameters and effective gas-phase densities. We compare our results to those in previous DMA studies as well as to ion diameters inferred from early and recent drift tube IMS-MS measurements in He [4,13,14,38]. Additionally, we demonstrate that the DMA-MS combination can distinguish between protein multimer ions formed nonspecifically during the electrospray process and those formed naturally in solution.…”

“…Tables 2 and 3 summarize inferred diameters from mobility measurements for cytochrome C and lysozyme ions, respectively, from this study and prior studies [4,13,14,38,63]. For each measured ion we list the number of protein monomers per ion and the charge state.…”

“…In all calculations the diameter of the gas molecule is accounted for; it is taken as 0.3 nm for "air" molecules [40] and 0.2 nm for He molecules [64]. Finally, the study to which each data point corresponds [4,13,14,38,63] and the ionization technique used in the study are listed. All other studies used He as a drift gas, and the ionization technique in each study was different (note that we perform ESI under nondenaturing conditions, while strongly denaturing acidic solutions were used in other studies).…”

“…The IMS-MS combination has recently been used for the identification of aggregates that are undetected by MS alone [2,3], the observation of coulombic stretching of highly charged protein ions [4,5], and to probe gas-phase protein ion structure [6][7][8][9][10][11][12][13][14]. Mobility measurements are particularly interesting in the case of high-mass protein ions generated by electrospray, which are capable of taking on different conformations in the gas phase.…”

An ion trap-ion mobility-time of flight mass spectrometer with three ion sources for ion/ion reactions

“…Varying the injection voltage and duration of the ion ejection pulse also induces similar changes in conformations of cytochrome c, especially the ϩ7 and ϩ8 ions [66]. We offer the following tentative, qualitative explanation for these observations.…”