Investigations of biological interactions by hydrogen deuterium exchange Fourier transform ion cyclotron mass spectrometry: novel methods, automated analysis and data reduction

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Blakney, Gregory Terrell

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Hydrogen/deuterium (H/D) exchange is used to investigate biological interactions by Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). For the first time, a series of oligonucleotides of varying length are interrogated by negative mode gas phase H/D exchange. Data presented describes the reactivity of these oligonucleotides and correlates reactivity to functional moieties of the model compound. The results of the study are subjected to center of mass analysis, a technique that uses the high mass resolving power of FT-ICR MS to facilitate an isotope-counting / abundance-weighted algorithm to determine deuterium incorporation. A maximum entropy method produces the relative reaction rates for each of the model compounds. Curve fitting of rates reveal >90% correlation between the areas of individual rate curves and available hydrogens. Observed data is consistent with literature reported reaction mechanisms. This work describes the successful implementation of the thorough high resolution analysis of spectra by Horn (THRASH) algorithm for the analysis of electron capture dissociation (ECD) spectra. Speed improvements in THRASH arise from optimized libraries and use of modern processors. The nonergodic nature of ECD alleviates deuterium scrambling and affords improved localization of exchange data. An ECD spectrum of ubiquitin is subjected to automated THRASH analysis resulting in 85% sequence coverage. Automated data analysis is extended to the batch processing of entire high performance liquid chromatography (HPLC) FT-ICR MS experiments. Center of mass calculations are determined by combining the data from multiple scans to maximize signal. Data is output as a single spreadsheet. Results of automated and manual data processing are compared. Complete analysis of a H/D exchange study is completed in two hours, rather than the two months required for manual analysis. Results based on this method are demonstrated for biological interactions of HIV capsid and Nop5-fibrilliarin complex.

New FT-ICR hardware increases data station throughput and reduces the amount of back exchange in liquid phase studies. The improved data station shows a three-fold improvement in scan speed and collects scans every 1.25 s. No significant loss of performance results from increased scan rates. Faster scan rates result in better chromatographic resolution and decreased spectral complexity.