TitleEnhanced Top-Down Protein Characterization with Electron Capture Dissociation and Cyclic Ion Mobility Spectrometry.
Publication TypeJournal Article
Year of Publication2022
AuthorsShaw JB, Cooper-Shepherd DA, Hewitt D, Wildgoose JL, Beckman JS, Langridge JI, Voinov VG
JournalAnal Chem
Date Published2022 Mar 08
KeywordsAmino Acid Sequence, Electrons, Ion Mobility Spectrometry, Proteins, Tandem Mass 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 (/ 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.

Alternate JournalAnal Chem
PubMed ID35188751
PubMed Central IDPMC8908312
Grant ListR43 GM123855 / GM / NIGMS NIH HHS / United States
R43 GM134792 / GM / NIGMS NIH HHS / United States
R44 GM123855 / GM / NIGMS NIH HHS / United States
R44 GM134792 / GM / NIGMS NIH HHS / United States