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Integration of tandem mass spectrometry and ion mobility spectrometry for protein characterization and structural analysis
Mass spectrometry (MS) based proteomics and intact protein analyses are important tools for the structural study of proteins and provide powerful methods for solving biochemical puzzles involving proteins. The work described in this dissertation is aimed at the development of novel, efficient, and information rich strategies for protein structure and sequence analysis. The approaches developed have been applied to analytes ranging from proteolytic peptides to large non-covalent protein complexes.^ Chapter I provides a comprehensive comparison of the MS approaches in existence in present day proteomics along with their implementation, suitability, and complementarity for effective proteome analysis. Ion dissociation methods, which serve as the foundation for tandem mass spectrometry (MS/MS) experiments, are next detailed in Chapter II. This chapter addresses the strengths and limitations of each ion dissociation method for MS/MS in light of the fundamentals of the dissociation processes involved. Mechanisms of peptide fragmentation chemistry and various combinations of these techniques with instrumental designs are also described. ^ MS/MS, despite being a broadly applied tool for protein characterization, often affords only a portion of the extractable information. Chapter III focuses on the orthogonal application of two complementary ion dissociation methods, viz. collision induced dissociation (CID) and electron transfer dissociation (ETD), coupled in conjunction with ion mobility spectrometry (IMS), a rapid gas-phase separation technique compatible with MS and MS/MS experiments. The approach has dual benefits of enhanced sequence coverage and more efficient use of the available ion population. The strategy is then escalated to a multidimensional protein complex analysis by merging the tools of native MS, top down MS/MS, and proteomics. Chapter IV demonstrates a method which combines multiple stages of CID bridged by IMS and targets non-covalent protein assemblies in a manner that allows subunits to be released and activated in a controlled stepwise fashion, to be separated based on their size and charge, and finally to yield subunit identities a single gas-phase analysis. In addition, IMS opens avenues for measurement of ion-neutral collisional cross sections of the analyte at each stage, allowing higher order structural analysis. This uniquely information-rich analysis method is the subject of Chapter V.^
Rathore, Deepali, "Integration of tandem mass spectrometry and ion mobility spectrometry for protein characterization and structural analysis" (2016). ETD collection for University of Nebraska - Lincoln. AAI10102681.