Fig. 2

GECX-MS to capture and identify affibody binding to Z in E. coli. a Structure of the affibody–Z protein complex (PDB ID 1LP1), with two proximal sites, Lys7 in the affibody and Glu24 in the Z protein, highlighted. b Western blot of cell lysate of cells co-expressing MPB-Z(24BprY) with affibody(7Cys) and of cells co-expressing MPB-Z(24EB3) with affibody(7Cys). The cross-linked MBP–Z/affibody complex is indicated with a star. c SDS-PAGE of proteins His-tag purified from cells co-expressing affibody(7Cys) with MPB-Z(24BprY), or with MBP-Z(24EB3). In the absence of Uaa, background suppression of the TAG codon at site 24 resulted in small amount of full-length MBP–Z. d Mass spectrum of the cross-linked peptide between affibody(7Cys) and MBP-Z(24BprY). U represents BprY in the peptide sequence. e His-tag purified affibody(7Cys)/MBP-Z(24EB3) was labeled with biotin via CuAAC and then western blotted with streptavidin-HRP conjugate. f Biotin based enrichment of cross-linked affibody(7Cys)/MBP-Z(24EB3). g Mass spectrum of the cross-linked peptide between affibody(7Cys) and MBP-Z(24EB3) before biotin labeling. U represents EB3 in the peptide sequence. h Mass spectrum of biotinylated cross-linked peptide between affibody(7Cys) and MBP-Z(24EB3). U represents EB3 in the peptide sequence. i Extracted ion chromatography of biotinylated cross-linked peptide from input sample of biotin enrichment. j Extracted ion chromatography of biotinylated cross-linked peptide from elute sample of biotin enrichment. RT retention time, MA peak area, MH peak height