Fig. 1: Incorporation of eFSY into proteins in E. coli with genetic code expansion and eFSY mediated cross-linking with cross-linked peptides enrichment. | Nature Communications

Fig. 1: Incorporation of eFSY into proteins in E. coli with genetic code expansion and eFSY mediated cross-linking with cross-linked peptides enrichment.

From: Characterize direct protein interactions with enrichable, cleavable and latent bioreactive unnatural amino acids

Fig. 1

a Chemical structure of eFSY. eFSY: enrichable fluorosulfate-L-tyrosine. (b) SDS-PAGE gel of His-tag purified MBP-Z(E24eFSY). c Western blot analysis showing MBP-Z(E24eFSY) labeled by biotin via click chemistry. CuAAC: Copper-catalyzed azide-alkyne cycloaddition. d Intact protein mass analysis of MBP-Z(E24eFSY). e Tandem mass spectrum of eFSY incorporated peptide of MBP-Z(E24eFSY) (‘U’ represents eFSY residue in this study, but not selenocysteine). f Tandem mass spectrum of a biotin-labeled peptide derived from MBP-Z(E24eFSY). g Proximal sites, Glu24 and Lys7, on the protein structure of affibody/Z complex (PDB ID 1LP1). Glu24 was used for eFSY incorporation, and Lys7 was used for target amino acid incorporation. (h) Western blot analysis showing cross-linking between MBP-Z(E24eFSY) and affibody(K7X). i Biotin labeling of cross-linked MBP-Z/affibody products. j MS/MS fragmentation patterns of cross-linked peptides and biotin-labeled peptides of MBP-Z/affibody cross-linking. k Extracted ion chromatograms (XICs) of cross-linked peptides from input sample (top panel: charge 3+ peptide precursor; bottom panel: charge 4+ peptide precursor). l XICs of cross-linked peptides from enriched sample, cross-linked peptides were effectively enriched by Monomeric Avidin beads (top panel: charge 3+ peptide precursor; bottom panel: charge 4+ peptide precursor). RT: retention time, MA: peak area. Source data are provided as a Source Data file.

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