Degree Type

Dissertation

Date of Award

2009

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Robert S. Houk

Abstract

As the widespread use of mass spectrometry (MS) for biological analyses,particularly in areas like proteomics and characterization of large macromolecular complexes, further improvements in MS instrumentation provide analytical capabilities that enable these new biological studies.

An ion trap ion trap - ion mobility - time of flight mass spectrometer with three ion sources was built which combines the capabilities of ion/ion reactions with ion mobility. The characterization of the instrument was demonstrated with ubiquitin. Ubiquitin ions from

either of two electrospray ionization (ESI) sources are stored in a 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 +7 ions are converted to lower charge states down to +1; the ions in lower charge states tend to be in compact conformations with cross sections down to ~ 880 y2. The duration and magnitude of the ion ejection pulse on the IT and the entrance voltage on the IM drift tube can affect the measured distribution of conformers for ubiquitin +7 and +6. 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.

Effects of ion/ion reactions on conformation of gas-phase cytochrome c were also studied on this instrument. Positive ions from cytochrome c are studied in a 3-D ion trap/ion vi

mobility (IM)/quadrupole-time-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. The conformation(s) of the product ions generated by ion/ion reactions in a given charge state are independent of the original charge state of the cytochrome c ions. In the lower charge states (+1 to +5) cytochrome c ions made

by the ion/ion reaction have a single conformation with cross section of ~ 1110 to 1180 y2, even if the original +8 ion started with two conformations. These cross section values are close to those of the "most folded" conformation found previously. In a given charge state, ions created by ion/ion reaction prefer to produce the more compact conformation in somewhat higher abundances, compared to those produced by the electrospray ionization (ESI) source alone. A variety of related studies that employ ion/ion reactions and IM to probe conformations of biomolecular ions should be possible by these methods.

A mass-selective ejection method from the ion trap was also developed. Instead of the applying a high voltage pulse to eject simultaneously, the ion trap mass-selective instability scan was used. Ions are ejected from the ion trap in a time dependent manner (sequentially): scanning the RF from 1 to 5 kV while a fixed frequency (47 kHz) sine wave was applied to the front cap of the ion trap with ramping amplitude from 4 V to 41 V. This ion trap ejection method provides another option for the separation of protein mixture ions, which have similar mobility but different mass to charge ratio. This capability has the potential to expand the ability of the ion mobility labeling parallel experiments to handle complex mixtures.

To improve the ion storage capacity and sensitivity of the ion trap instrument, a linear ion trap (LIT) with electrospray ionization (ESI) has been constructed for ion/ion reactions. To reduce the instrument's complexity and make it available for wide dissemination, only a few simple electronics components were custom built. The instrument functions as both a reaction vessel for gas-phase ion/ion reactions, and a mass spectrometer using mass-selective axial ejection. Initial results demonstrate trapping efficiency of 70 to 90% and the ability to perform proton transfer reactions on intact protein ions, including dual polarity storage reactions, transmission mode reactions, and ion parking.

DOI

https://doi.org/10.31274/etd-180810-2666

Copyright Owner

Qin Zhao

Language

en

Date Available

2012-04-28

File Format

application/pdf

File Size

169 pages

Included in

Chemistry Commons

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