Chemical strategies for profiling protein-protein interactions and protein posttranslational modifications

Chemical tools are becoming very popular in the field of mass spectrometry based proteomics. In this dissertation, I present my work on novel analytical strategies that use chemical properties of the specific amino-acids in proteins to profile protein-protein interactions and protein posttranslational modifications.; A solid-phase phosphopeptide enrichment strategy was developed to reduce the sample complexity before mass spectrometric identification. Modification of the phosphopeptides was achieved by a base-catalyzed elimination and Michael addition chemical strategy. Selective enrichment was achieved with a maleimide-bound acid-cleavable solid-support reagent.; Three novel chemical cross-linkers, called protein interaction reporters (PIRs), were developed introducing several chemical features in the cross-linker backbones. These cross-linkers have mass spectrometry identifiable features. They are constructed with two mass spectrometry cleavable-bonds. In the gas phase in the mass spectrometer, they generate reporter ions from the cross-linked peptides. This reporter ion strategy has the ability to distinguish dead-end, intra- and inter-cross-linked peptides. My research demonstrated gas phase fragmentation of these PIRs and PIR-labeled cross-linked peptides.; The gas phase fragmentation of mono-oxidized thio-ether residues (sulfoxides) in the peptides was also investigated. This oxidative modification occurs during proteomics sample preparations in the modified cysteinyl side-chains. We demonstrated fragmentation of these side-chain containing oxidized thio-ether residues utilizing low energy collisions and electron capture dissociations. The fragmentation of oxidized thio-ether within the peptide sequence was also investigated and mechanism was proposed for the gas fragmentation behavior of these modification-containing peptides.; A chemical strategy was also developed for selective isolation of N-terminal peptides utilizing guanidination of lysine residues in the proteins. In this regard, a mass spectrometric study on the tryptic hydrolysis of the homoarginine modified proteins was also performed. Before enzymatic digestion, the original N-termini of proteins were blocked by an acetylation reaction. After enzymatic digestion, the newly formed N-termini were captured with a solid-support reagent, leaving original blocked N-termini in the solution.; In this dissertation, four novel mass spectrometry based chemical strategies were demonstrated to profile protein posttranslational modifications and protein-protein interactions. We believe these studies will significantly contribute in the field of proteomics.