Sponsor
Chemistry
Chemistry Defense: Yueqi Chen:"Mass spectrometry-based proteomic approach for thermodynamic analysis of both global and local protein unfolding"
Wednesday, July 15,
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Mass spectrometry-based proteomic approach for thermodynamic analysis of both global and local protein unfolding
Mass spectrometry-based protein stability measurements have become powerful tools for analyzing protein folding, ligand binding, and conformational changes in biological mixtures. Established approaches, including stability of proteins from rates of hydrogen-deuterium exchange (SUPREX), stability of proteins from rates of oxidation (SPROX), pulse proteolysis (PP), and thermal proteome profiling (TPP), have enabled proteome-scale measurements of stability changes.
In this dissertation, established stability-based proteomic methods, including SPROX-, LiP-, and TPP-based approaches, were first used to characterize drug-induced stability and expression-level changes in Plasmodium heat shock proteins. These studies provided useful information about selected compounds with Plasmodium Hsp70 and Hsp90, but also highlighted limitations of existing approaches, especially when only small fold changes were observed or when established strategies provided incomplete thermodynamic information. These methods primarily report global protein unfolding behavior, limiting their ability to resolve local structural fluctuations and other native-state information.
To address this problem, this dissertation develops native-state thermodynamic analysis using mass spectrometry (NSTA-MS), a new approach for measuring global and local protein folding thermodynamics in complex proteomes. The method uses denaturant- and time-dependent labeling of selected residues, followed by quantitative mass spectrometry and a new data analysis strategy. This strategy enables the determination of global unfolding free energies, local unfolding free energies, native-state protection, and apparent pKa values of cysteine thiols.
The method is demonstrated using both Cys and Met labeling strategies. The Cys labeling approach was applied to yeast and MCF-7 cell lysates, where NSTA-MS assayed 1,096 and 3,703 cysteines from 647 and 1,813 proteins, respectively. The Met labeling strategy identified 1,511 methionines from 725 proteins in yeast lysate. Together, these strategies provided complementary structural information and increased protein and peptide coverage.
Overall, this dissertation establishes NSTA-MS as a thermodynamic analysis method for probing global and local protein folding in complex proteomes.
Mass spectrometry-based protein stability measurements have become powerful tools for analyzing protein folding, ligand binding, and conformational changes in biological mixtures. Established approaches, including stability of proteins from rates of hydrogen-deuterium exchange (SUPREX), stability of proteins from rates of oxidation (SPROX), pulse proteolysis (PP), and thermal proteome profiling (TPP), have enabled proteome-scale measurements of stability changes.
In this dissertation, established stability-based proteomic methods, including SPROX-, LiP-, and TPP-based approaches, were first used to characterize drug-induced stability and expression-level changes in Plasmodium heat shock proteins. These studies provided useful information about selected compounds with Plasmodium Hsp70 and Hsp90, but also highlighted limitations of existing approaches, especially when only small fold changes were observed or when established strategies provided incomplete thermodynamic information. These methods primarily report global protein unfolding behavior, limiting their ability to resolve local structural fluctuations and other native-state information.
To address this problem, this dissertation develops native-state thermodynamic analysis using mass spectrometry (NSTA-MS), a new approach for measuring global and local protein folding thermodynamics in complex proteomes. The method uses denaturant- and time-dependent labeling of selected residues, followed by quantitative mass spectrometry and a new data analysis strategy. This strategy enables the determination of global unfolding free energies, local unfolding free energies, native-state protection, and apparent pKa values of cysteine thiols.
The method is demonstrated using both Cys and Met labeling strategies. The Cys labeling approach was applied to yeast and MCF-7 cell lysates, where NSTA-MS assayed 1,096 and 3,703 cysteines from 647 and 1,813 proteins, respectively. The Met labeling strategy identified 1,511 methionines from 725 proteins in yeast lysate. Together, these strategies provided complementary structural information and increased protein and peptide coverage.
Overall, this dissertation establishes NSTA-MS as a thermodynamic analysis method for probing global and local protein folding in complex proteomes.
