Investigating protein surfaces and protein interactions using the element-coded affinity tags (ECAT).

摘要

Proteomics is the study of a cell's protein population in a given state. The challenges in proteomics include the large dynamic range of the various proteins, the post-translational modifications such as phosphorylation and oxidation, and the solubility issues with membrane proteins and the like.; Here we present new techniques that utilize Element-Coded Affinity Tags (ECAT). ECAT are comprised of a reactive group and a DOTA metal-chelating moiety loaded with a rare earth. The ECAT system has a number of advantages over current isotope tags. First, a variety of reactive groups are available: thiol for cysteine residues, aminooxy for carbonyl groups, and isothiocyanate for lysine residues. Second, there are seven monoisotopic rare earths so ECAT can be multiplexed up to seven-fold with the same tag. Third, the ECAT system co-elutes in reverse-phase chromatography. Finally, the monoclonal antibody against M-DOTA, 2D 12.5, can be used to detect, quantify, and purify ECAT-labeled species. The quantification occurs both at protein and individual peptide levels.; This dissertation discusses how the ECAT system can be used to map protein surfaces and how it can be used with FeBABE (iron (III) (S)-1-[ p-(bromoacetamido)benzyl]ethylenediaminetetraacetic acid) to map protein:protein interactions. First, we show that affinity purification of ECAT-peptides using the 2D12.5-aminolink system can be improved with the addition of detergents, specifically Triton X-100. Next, we demonstrate the utility of an oxidation-specific ECAT, ((S)-2-(4-(2-aminooxy)-acetamido)-benzyl)-DOTA (AOD) to map the surface of human serum albumin. This is the first example of using an oxidation-specific tag to map several types of oxidized amino acids and the first example quantifying oxidation at both the protein and peptide levels. AOD is combined with FeBABE to generate a system of site-specific oxidation followed by ECAT detection and quantitation. This is validated using lysozyme and two of its monoclonal antibodies, D1.3 and D44.1 and the interaction between albumin and calmodulin. Another ECAT, (S)-2-(4-isothiocyanatobenzyl)-DOTA (DITC) is used to study cell-surface proteins on the Raji cancer cell line. Overall, this dissertation seeks to highlight the many benefits of the ECAT system and to showcase its utility with specific examples.