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

Abstract

This instrument combines the capabilities of ion/ion reactions with ion mobility (IM) and time-of-flight (TOF) measurements for conformation studies and top-down analysis of large biomolecules. Ubiquitin ions from either of two electrospray ionization (ESI) sources are stored in a three dimensional (3D) ion trap (IT) and reacted with negative ions from atmospheric sampling glow discharge ionization (ASGDI). The proton transfer reaction products are then separated by IM and analyzed via a TOF mass analyzer. In this way, ubiquitin ϩ7 ions are converted to lower charge states down to ϩ1; the ions in lower charge states tend to be in compact conformations with cross sections down to ϳ880 Å 2 . The duration and magnitude of the ion ejection pulse on the IT exit and the entrance voltage on the IM drift tube can affect the measured distribution of conformers for ubiquitin ϩ7 and ϩ6. Alternatively, protein ions are fragmented by collision-induced dissociation (CID) in the IT, followed by ion/ion reactions to reduce the charge states of the CID product ions, thus simplifying assignment of charge states and fragments using the mobility-resolved tandem mass spectrum. Instrument characteristics and the use of a new ion trap controller and software modifications to control the entire instrument are described. Ion mobility (IM) [4,5] has become a very useful technique for analysis of biological ions in the gas phase [6]. IM provides information about ion size and structure [7], as it rapidly separates ions based on collision cross-section, rather than just m/z ratio. The use of IM to disperse a mixture of ions in time before analysis via a time-of-flight (TOF) MS, i.e., nested drift (flight) time measurements, is an important recent advance. These experiments were pioneered by Clemmer and coworkers in the mid-1990s [8], and have now been used by several other groups [9 -13]. The recent Synapt ESI-IMS-MS by Waters Corp. provides a commercially available instrument for gas-phase ion conformation studies by IM using a novel traveling wave approach [9].In a series of instrumental designs, the Clemmer group has made various modifications to the initial ESI-IM-TOF, including the insertion of a collision cell between the IM drift tube and the TOF for mobility labeling experiments [14,15], and the addition of an IT before the mobility drift tube to improve the duty cycle from the continuous ESI source [16,17]. One publication demonstrated MS/MS capabilities with an ion trap before IM-TOF [18], but the entire instrument was not under computer control. Therefore, only relatively simple experiments were possible.IM has been used to analyze the products of ionmolecule reactions [19], including proton transfer [20,21], H/D exchange [22], and solvation [23][24][25][26]. In these studies, the desired reactions take place either in the atmospheric pressure ion source interface region or in the drift tube itself. Thus, only short reaction times and certain reagent ions can be used. In addition, performing reactions in the IM cell can make spe...

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

“…The design and general operating conditions for the home-built three-source instrument are described in another paper [26]. Bovine heart cytochrome c (SigmaAldrich, St. Louis, MO, USA) was used without further purification in all experiments.…”

“…The default operating conditions for ion mobility measurement were as follows. The ion trap ejection pulse was zero to Ϫ100 V for 3 s, and the injection voltage on the drift tube entrance was Ϫ50 V. These conditions provided the best signal and minimized variation of IM spectra with duration of the injection pulse [26]. The injection voltage was varied for Figure 1, and Ϫ50 V was used for Figure 6, for reasons discussed below.…”

“…Cross-section distributions for cytochrome c 8ϩ ions at different injection voltage into the drift tube. resolved mobility spectra as described previously [3,26]. In particular, the drift time is calculated by measuring the total time between the ion trap exit pulse and the observation of ions at the TOF detector.…”

“…In particular, the drift time is calculated by measuring the total time between the ion trap exit pulse and the observation of ions at the TOF detector. The flight time between the exit of the drift tube and the TOF source region is calculated from the instrument dimensions, applied voltages and m/z ratio and subtracted from the total time to give the actual time spent in the drift tube [26].…”

“…A three-source-ion trap-drift tube-q-TOF instrument was recently developed by our group to combine ion/ ion reactions with IM-TOF measurements [26,27]. The present paper describes how proton transfer ion/ion reactions can be used to manipulate charge state and study the effect of ion/ion reactions on the conformations of cytochrome c ions.…”