Engineered Single-Domain Antibodies with High Protease Resistance and Thermal Stability
Figure 2
Disulfide bond formation between residues Cys54 and Cys78 is confirmed by MS2.
(A) Schematic diagram of mutant VHH digestion with cyanogen bromide (CNBr) and trypsin before MS2 analysis. (B) VHHs (3 µg per lane) were subjected to SDS-PAGE analysis under non-reducing (NR) conditions to illustrate near complete digestion with CNBr and trypsin. Untreated A5.1m was added as a control (Ctl). M: molecular weight marker in kDa. (C) MaxEnt 3 deconvoluted CID-MS2 spectrum of the m/z 526.25 (3+) ion of the disulfide-linked peptide EFVCVITR (P1) – FTCSR (P2), encompassing the Cys54–Cys78 disulfide bond, from CNBr/trypsin digested A5.1m.