Fig. 4.

Functionalization of proteins with azides, biotin and PEG. a Schematic representation of the two-step biotinylation of GH₆-EGFP. Reaction conditions for the biotinylation step: 100 µM azido-functionalized N₃-CH₂-C(O)-GH₆-EGFP with 1.5 equiv. of 19 in 100 mM phosphate buffer with 1% acetonitrile at pH 7.5 and room temperature for 1 h. b Deconvoluted ESI-TOF spectrum of the reaction of GH₆-EGFP with phenyl ester 18. c Deconvoluted ESI-TOF spectrum of the reaction of azido-functionalized GH₆-EGFP with DBCO-PEG4-biotin 19. d SDS-PAGE analysis of the two-step PEGylation of GH₆-EGFP. Lane 1: protein marker, lane 2: GH₆-EGFP, lane 3: azido-functionalized GH₆-EGFP, lane 4: reaction of azido-functionalized GH₆-EGFP with 5 kDa DBCO-PEG. Reaction conditions for the PEGylation step: 100 µM azido-functionalized N₃-CH₂-C(O)-GH₆-EGFP with 2.5 equiv. of 5 kDa DBCO-PEG in 100 mM phosphate buffer at pH 7.5 and room temperature for 3 h. e Deconvoluted ESI-TOF spectra of the reactions of GH₆-SUMO (Supplementary Table 1, Entry 8) and GH₆-MBP with phenyl ester 18. Unmodified proteins are labeled SM, and species corresponding to the product masses are labeled P1 and P2 (the number indicating the number of azides or biotins, respectively)