Summary
This project aims to improve fluorosequencing technology for parallel, single-molecule peptide/protein analysis by addressing challenges in sequential and selective amino acid labeling, differentiating N- and C-terminal residues, and extending the method to post-translational modifications.
What they want
The project focuses on overcoming the lack of approaches for sequential and selectively labeling multiple amino acids on the same peptide/protein with different tags, as well as differentiating N-terminal and C-terminal residues from lysine and Glu/Asp AAs. It will explore labeling four specific amino acids (Cys, Lys, Tyr, Typ, His, Ser, Thr, Glu/Asp, Arg, PSer, PThr, PTyr) and extending fluorosequencing to post-translational modifications like mono, di, and trimethylated Lys and ubiquitination. This involves using 'click:clack' pairs with four fluorophores for TIRF microscopy. The project will also address FRET issues by exploring pH-controlled fluorophores and develop a base-induced method for N-terminal chain-end sequencing to overcome the instability of common fluorophores during TFA treatment in Edman degradation.
Technical requirements
- TIRF 4-channel microscope
- Classic Edman degradation
- Fluorophore labeling of amino acids
- Conjugation handles carrying 'click' partners within 'click:clack' pairs
- Designed fluorophores whose emission can be turned on and off by intramolecular conjugate additions controlled by varying pH
- Base-induced method for N-terminal chain-end sequencing
