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Mass Spectrometry-Based Elucidation of Protein Structure Using Noncovalent Molecular Recognition and Photo-Induced Radical Chemistry


Advances in mass spectrometry (MS) have enabled quick and accurate protein identification, an important analytical goal. However, knowledge of the identity of a protein only skims the surface of the structural and functional complexities that are characteristic of proteins. Described in this dissertation is the development of MS-based analytical techniques to investigate the structure of proteins using noncovalent interactions and photo-induced radical chemistry.

The first half of the dissertation reports the discovery of a novel gas phase protein dissociation method using photo-induced radical chemistry to `direct' backbone fragmentation to specific amino acid residues, which we have called `radical directed dissociation' or RDD. Residue-specific dissociation, which is nearly unprecedented in the literature, is a significant advance towards enzyme-like protein disassembly in the gas phase. Investigation of numerous peptides and proteins yields two key features of RDD. First, fragmentation frequently occurs at tyrosine, phenylalanine, tryptophan, histidine, threonine, and serine. These residues have the lowest predicted C(beta)-H bond dissociation energies (BDEs) of the twenty canonical amino acids. Second, removal of the beta carbon of tyrosine abrogates backbone fragmentation at that residue. These results indicate that the C(beta) is the most important site for radical-induced backbone fragmentation and that radical migration in peptides appears to favor sites with the lowest C-H BDEs. We have called this latter phenomenon "radical funneling". Although useful for peptides, the "radical funneling" model becomes inappropriate for larger peptides and proteins, where structural effects cannot be ignored. Indeed, we show that radical migration is a sensitive reporter of tertiary structure of proteins in vacuo.

The remaining half of the dissertation reports the development of selective noncovalent adduct protein probing mass spectrometry (SNAPP-MS) and radical migration as methods of investigating the three-dimensional structures of proteins. Information on three-dimensional structure is typically unobtainable by MS. However, orthogonal techniques may be used to translate structural information into changes that are observable by MS. SNAPP-MS is a fast technique that uses 18-crown-6 ether (18C6) as a molecular probe of lysine availability in proteins. The number of 18C6s that bind to protein is easily measured by MS. Interestingly, the binding of 18C6 to protein occurs in solution, which allows investigation of solution phase structure.

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