Ion-Ion Chemistry and Parallel Ion Parking for Advances in Mass Spectrometric Analysis of Intact Proteins

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Ugrin, Scott, Chemistry - Graduate School of Arts and Sciences, University of Virginia
Ugrin, Scott, Arts & Sciences Graduate-uasg, University of Virginia

Mass spectrometry (MS) is considered the preferred tool for the structural characterization of proteins. Typical protein studies by MS involve breaking proteins into small pieces prior to analysis. Recently, there has been a concerted effort in the field of protein mass spectrometry to enable analyses of proteins in their intact form. This type of analysis allows for the ability to differentiate between closely related species, a feature not offered by peptide-based methods. Ion-ion chemistry, namely electron transfer dissociation (ETD) and ion-ion proton transfer (IIPT), has become a staple in interrogating the primary structure of intact proteins. However, detection of whole proteins using traditional mass spectrometry methods has proven difficult given their high molecular weight. The ideal MS method would allow for the identification of all proteins in a sample mixture. This includes the characterization of the entire amino acid sequence and the site-localization of all post-translational modifications (PTMs) on each protein species. However, low signal-to-noise ratios of intact proteins in both the full mass spectra and tandem mass spectra (MS/MS) limit our ability to perform this type of analysis.
Here we describe the implementation and application of novel MS methods for enhancing the signal-to-noise ratio of intact proteins and their component fragment ions. We accomplish this by selectively controlling the kinetics of products of gas phase ion-ion reactions using a technique called parallel ion parking. Using these methods we demonstrate near-complete sequence coverage of the intact protein apomyoglobin (17 kDa). In addition, we are able to preserve large fragment ions during ETD of the light chain of a monoclonal antibody (25 kDa). Further, we introduce parallel ion parking during IIPT which allows us to concentrate the signal of several unknown proteins concurrently. We use this technique to develop an automated method of identifying intact proteins in a mixture, which range in mass from 4.5 to 61 kDa. We accomplish this on a chromatographic time-scale using IIPT and data-dependent higher energy collisionally activated dissociation (HCD).

PHD (Doctor of Philosophy)
Mass Spectrometry, Electron Transfer Dissociation, Ion-Ion Proton Transfer, Intact Protein MS, Proteomics, Parallel Ion Parking
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