Synthetic chemical approaches to proteomics: Affinity labeling and protein functional group modification.

摘要

Target identification probe (TIP) reagents have been designed for the determination of the protein targets of biologically active small molecules through photoaffinity labeling. These reagents combine photoaffinity, affinity (biotin), and stable mixed isotope labels. The mixed isotope label serves as a selective identification tag on mass spectrometry to clearly indicate photolabeled species in the presence of contaminants which are common to these approaches. The utility of the technique was demonstrated by the identification of cyclophilin A as the target of the small molecule ligand cyclosporin A from within a protein mixture. Improved reagents were synthesized containing several different photocrosslinking moieties and a chemically orthogonal alkyne tag for improved purification via "click" chemistry. The crosslinking efficiency of cyclosporin A TIP conjugates to cyclophilin A was evaluated.; A method was developed for the site-specific, covalent modification of fusion proteins by affinity labeling with an engineered receptor-ligand pair. The introduction of thiol and acrylamide groups in the receptor and ligand, respectively, enables the formation of a covalent complex by a proximity-accelerated reaction. Using the cyclophilin-cyclosporin receptor-ligand pair, the technique was used to selectively label a fusion protein in live cells with a small molecule fluorophore. The modular approach described should facilitate the development of many new protein-small molecule tagging systems.; A sequential reaction methodology for the complete derivatization of protein thiols, amines, and acids in high purity under denaturing conditions has been developed. Following standard thiol alkylation, protein amines are modified via reductive methylation with formaldehyde and pyridine-borane. Protein acids are subsequently amidated with amines using a peptide coupling reagent under buffered conditions in dimethylsulfoxide. The generality of the approach was demonstrated with four proteins and with several amines yielding near quantitative transformations as characterized by high resolution Fourier Transform mass spectrometry. The developed chemistry has numerous applications in mass spectrometry based proteomics of intact proteins, including the addition of stable isotopes for relative quantitation, protein identification through functional group counting, and mechanistic investigations into protein charging in mass spectrometry. Additionally, this chemistry has implications for peptide modifications and in the semi-synthesis of monodisperse polymers based on protein scaffolds.