Enhanced Top-Down Protein Characterization with Electron Capture Dissociation and Cyclic Ion Mobility Spectrometry.

Tandem mass spectrometry of denatured, multiply charged high mass protein precursor ions yield extremely dense spectra with hundreds of broad and overlapping product ion isotopic distributions of differing charge states that yield an elevated baseline of unresolved "noise" centered about the precursor ion. Development of mass analyzers and signal processing methods to increase mass resolving power and manipulation of precursor and product ion charge through solution additives or ion-ion reactions have been thoroughly explored as solutions to spectral congestion. Here, we demonstrate the utility of electron capture dissociation (ECD) coupled with high-resolution cyclic ion mobility spectrometry (cIMS) to greatly increase top-down protein characterization capabilities. Congestion of protein ECD spectra was reduced using cIMS of the ECD product ions and "mobility fractions", that is, extracted mass spectra for segments of the 2D mobiligram (m/z versus drift time). For small proteins, such as ubiquitin (8.6 kDa), where mass resolving power was not the limiting factor for characterization, pre-IMS ECD and mobility fractions did not significantly increase protein sequence coverage, but an increase in the number of identified product ions was observed. However, a dramatic increase in performance, measured by protein sequence coverage, was observed for larger and more highly charged species, such as the +35 charge state of carbonic anhydrase (29 kDa). Pre-IMS ECD combined with mobility fractions yielded a 135% increase in the number of annotated isotope clusters and a 75% increase in unique product ions compared to processing without using the IMS dimension. These results yielded 89% sequence coverage for carbonic anhydrase.

Applications of a travelling wave-based radio-frequency-only stacked ring ion guide.

The use of radio-frequency (RF)-only ion guides for efficient transport of ions through regions of a mass spectrometer where the background gas pressure is relatively high is widespread in present instrumentation. Whilst multiple collisions between ions and the background gas can be beneficial, for example in inducing fragmentation and/or decreasing the spread in ion energies, the resultant reduction of ion axial velocity can be detrimental in modes of operation where a rapidly changing influx of ions to the gas-filled ion guide needs to be reproduced at the exit. In general, the RF-only ion guides presently in use are based on multipole rod sets. Here we report investigations into a new mode of ion propulsion within an RF ion guide based on a stack of ring electrodes. Ion propulsion is produced by superimposing a voltage pulse on the confining RF of an electrode and then moving the pulse to an adjacent electrode and so on along the guide to provide a travelling voltage wave on which the ions can surf. Through appropriate choice of the travelling wave pulse height, velocity and gas pressure it will be shown that the stacked ring ion guide with the travelling wave is effective as a collision cell in a tandem mass spectrometer where fast mass scanning or switching is required, as an ion mobility separator at pressures around 0.2 mbar, as an ion delivery device for enhancement of duty cycle on an orthogonal acceleration time-of-flight (oa-TOF) mass analyser, and as an ion fragmentation device at higher wave velocities.