Proteomic Methods and Applications for Protein Folding Stability Measurements

In the last 10 years, several mass spectrometry-based proteomic techniques have been developed for the large-scale characterization of protein conformations, thermodynamic stabilities, and protein−ligand interactions. The main focus of this dissertation involves the development and application of several mass spectrometry-based-methods in this current suite of proteomics techniques for the large-scale analysis of protein folding and stability measurement. One goal of this work is to investigate the use of protein folding and stability measurements to better detect and understand the biophysical properties of post-translational modifications. Another goal of this work is to develop a novel protein stability measurement technique for making thermodynamic measurements of protein folding and ligand binding interactions. This technique, which involves a combination chemical denaturant and protein precipitation yields significantly better proteomic coverage and a largely reduced false discovery rate compared to its sister technique, Stability of Proteins from Rates of Oxidation (SPROX).

The first part of this dissertation describes the application of the stability of proteins from rates of oxidation (SPROX) and limited proteolysis (LiP) on comparing the conformational properties of proteins in two MCF-7 cell lysates including one that was and one that was not dephosphorylated with alkaline phosphatase. A total of 168 and 251 protein hits were identified with dephosphorylation-induced stability changes using the SPROX and LiP techniques, respectively. The SPROX results revealed that the magnitudes of the destabilizing effects of dephosphorylation on the different aaRSs were directly correlated with their previously reported aminoacylation activity change upon dephosphorylation. The example of these aaRSs substantiates the close link between protein folding thermodynamic and function and helps establish the utility of thermodynamic stability measurements for understanding protein function.

The second part of this dissertation describes the development of a new protein-stability based proteomic method for identification and quantification of protein-drug interactions. The approach involves the evaluation of ligand-induced protein folding free energy changes (ΔΔGf) using chemical denaturation and protein precipitation (CPP) to identify the protein targets of drugs and to quantify protein−drug binding affinities. In the proof-of-principle studies performed here, the CPP technique was able to identify the well-known protein targets of cyclosporin A and geldanamycin in a yeast lysate. The technique was also used to identify protein targets of sinefungin in a human MCF-7 cell lysate. The CPP technique yielded dissociation constant (Kd) measurements for these well-studied drugs that were in general agreement with previously reported Kd or IC50 values.

The third part of this dissertation describes two protein target discovery applications of the CPP approach including one involving subglutinol A (a natural product with immunosuppressive activity) and one involving clemastine fumarate (an existing anti-histamine drug with recently discovered anti-malarial activity). As part of this work, about 800 proteins in a mouse 2B4 T cell lysate were assayed for subglutinol A-induced stability changes, and deoxycytidine kinase was identified as the protein hit. The magnitude of the ligand induced stability change was used to calculate a Kd value of 250 M, which is close to the reported cell based IC50. In the protein target discovery study on clemastine fumarate, a total of 800 yeast proteins were assayed for drug-induced stability changes and 8 protein with clemastine-induced stability changes were identified, including SEC14 cytosolic factor, glycerol kinase, asparagine--tRNA ligase, and inosine triphosphate pyrophosphatase. The latter two applications demonstrate that CPP can reliably identify and quantify protein-drug interactions in a complex biological mixture, making it a valuable addition to the current suite of proteomic methods for the large-scale detection and quantitation of protein-ligand binding interactions.