Extended Data Fig. 4: Testing Trp-CLiC on peptides and proteins.
From: Oxidative cyclization reagents reveal tryptophan cation–π interactions
a-c) Measuring spectroscopic changes and reaction kinetics between oxaziridine and tryptophan. a) The time-dependent UV-Vis absorbance of Trp-CLiC reaction between Ac-Trp-OMe (100 μM) with Ox-W18 (100 μM). As reaction proceeded, the spectroscopic changes showed the absorbance decreases at 280 nm and the intensity increases at 245 nm. b) The A260/A280 ratio, which could be easily monitored via NanoDrop spectrophotometer, could be utilized to calculate the extent of the Trp-CLiC reaction process. c) After obtaining the observed rate constants k’ under different concentrations of Ac-Trp-OMe (500 μM, 550 μM, and 600 μM), the second-order rate constant could be determined by the slope of the k’ against the Ac-Trp-OMe concentrations. d) Scheme of tryptophan modification on GLP-1. e) Crystal structure of IL8 (PDB: 2il8) featuring one native buried tryptophan residue in stick form. f) IL8 could be labelled after denaturing to expose the tryptophan residue. Results are representative of two biological replicates. g) LC-MS/MS analysis further confirmed the site-specific bioconjugation of tryptophan residue on IL8 protein. The signal peaks marked with asterisks represent the peptide products after MS cleavage and neutral loss. h) In-gel fluorescence imaging showed that oxidative cyclization reaction on BSA tryptophan residues was rapid. i) The denaturing conditions triggered an increase in the labelled tryptophan sites of Lysozyme by Trp-CLiC. Numbers of Ox-W18 targeting on representative amino acids, namely Trp, Met, Lys, Cys, Tyr, and His confirmed the specific targeting on tryptophan. Results are representative of two biological replicates. j) LC-MS/MS analysis showed that W62, the most surface-exposed site of Lysozyme, could be labelled by Trp-CLiC under native folded conditions, whereas the adjacent but more buried site W63 could only be targeted after protein denaturation. The signal peaks marked with asterisks represent the peptide products after MS cleavage and neutral loss.
