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. Author manuscript; available in PMC: 2020 Apr 17.
Published in final edited form as: J Am Chem Soc. 2019 Apr 8;141(15):6204–6212. doi: 10.1021/jacs.8b12954

Figure 5.

Figure 5.

Site-specific double labeling of anti-HER2 Fab using CRACR and SPAAC. A) The Fab was expressed incorporating 5HTP and pAzF into 169 and 202 sites of the heavy chain using the dual-ncAA incorporation system described in Figure 4. B) ESI-MS analysis of Fab-169-5HTP-202-pAzF (not reduced; HC and LC connected by a disulfide) shows the expected mass (top panel).Treatment of this Fab double mutant with 4CDZ or DBCO-tamra produces expected single modification, while quantitative double modification is observed when both are used. Identical treatment of the wild-type Fab leads to no modification (Supplementary Figure S4). C) Labeling Fab-169-5HTP-202-pAzF or wild-type Fab with DBCO-tamra or fluorescein-diazonium or both leads to expected labeling of the double mutant but not the wild-type, as revealed by reducing SDS-PAGE (HC and LC separates; each ~24 kDa) followed by fluorescence imaging.