Fourier Transform Analysis for the Characterization of Mass Heterogeneity of Intact Protein Complexes Using Native Mass Spectrometry

Abstract

Heterogeneous mass populations, or molecules that have molecular weight distributions for their mass measurement, appear in many contexts throughout chemistry, including multi-subunit protein complexes, lipid-bound membrane proteins, and polymers. The average mass and dispersity of these molecules are useful and pertinent parameters to measure and investigate, as these properties can have dramatic effects on the physical properties of the overall system. Mass spectrometry has emerged as a powerful tool to probe these parameters, but conventional mass spectrometry can be problematic for heterogeneous biological samples, as the experiment requires transfer to the gas phase and can sometimes require harsh ionization conditions. Native mass spectrometry can overcome these limitations, in that it can maintain the native stoichiometry and structure of biomolecular complexes into the gas phase, but as the dispersity and size of these molecules increases, it can become increasingly difficult to measure the average mass and dispersity due to mass spectral congestion. This congestion of peaks can often obfuscate determination of charge state, total mass, or subunit mass using conventional mass spectrometry analysis methods.

Here, research is presented dedicated to the development of a Fourier transform-based method that can be used to deconvolve highly congested mass spectra for a variety of different heterogeneous mass populations. The method is parameter-free and requires no initial guesses of charge states, total mass, or subunit mass, thus giving it a unique advantage over other established techniques. First, a 1-dimensional Fourier analysis is introduced that can probe the subunit mass, charge states, and subunit mass dispersity for a variety of different molecules. The method is further advanced by discussing the advantages of using higher harmonic frequencies in the Fourier spectrum, particularly for mass spectra with low signal-to-noise and poor resolution. A short-time Fourier transform-based method is then introduced, and is demonstrated to be useful for extracting signal from native-like protein ions even in the presence of a large salt-cluster background. Finally, the theoretical and practical implications for investigating mass populations with two or more different subunits is explored. This dissertation includes previously published co-authored material.