Investigations of biological interactions by hydrogen deuterium exchange Fourier transform ion cyclotron mass spectrometry: novel methods, automated analysis and data reduction
Year: 2003
Type: dissertation
Abstract: Hydrogen/deuterium (H/D) exchange is used to investigate biological \ninteractions by Fourier transform ion cyclotron resonance mass spectrometry (FTICR \nMS). For the first time, a series of oligonucleotides of varying length are \ninterrogated by negative mode gas phase H/D exchange. Data presented describes \nthe reactivity of these oligonucleotides and correlates reactivity to functional \nmoieties of the model compound. The results of the study are subjected to center of \nmass analysis, a technique that uses the high mass resolving power of FT-ICR MS \nto facilitate an isotope-counting / abundance-weighted algorithm to determine \ndeuterium incorporation. A maximum entropy method produces the relative reaction \nrates for each of the model compounds. Curve fitting of rates reveal >90% \ncorrelation between the areas of individual rate curves and available hydrogens. \nObserved data is consistent with literature reported reaction mechanisms. \n This work describes the successful implementation of the thorough high \nresolution analysis of spectra by Horn (THRASH) algorithm for the analysis of \nelectron capture dissociation (ECD) spectra. Speed improvements in THRASH arise \nfrom optimized libraries and use of modern processors. The nonergodic nature of \nECD alleviates deuterium scrambling and affords improved localization of \nexchange data. An ECD spectrum of ubiquitin is subjected to automated THRASH \nanalysis resulting in 85% sequence coverage. \n Automated data analysis is extended to the batch processing of entire high \nperformance liquid chromatography (HPLC) FT-ICR MS experiments. Center of \nmass calculations are determined by combining the data from multiple scans to \nmaximize signal. Data is output as a single spreadsheet. Results of automated and \nmanual data processing are compared. Complete analysis of a H/D exchange study \nis completed in two hours, rather than the two months required for manual analysis. \nResults based on this method are demonstrated for biological interactions of HIV \ncapsid and Nop5-fibrilliarin complex. \n \n New FT-ICR hardware increases data station throughput and reduces the \namount of back exchange in liquid phase studies. The improved data station shows a \nthree-fold improvement in scan speed and collects scans every 1.25 s. No significant \nloss of performance results from increased scan rates. Faster scan rates result in \nbetter chromatographic resolution and decreased spectral complexity.
Author: Greg T. Blakney
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W1862388118