High yield of cysteine derivatization after L19 IgG-GyrA in vitro cleavage. Purified EPL product was pepsin digested. (A) Scheme indicating observed pepsin cleavage sites. (B) SDS-PAGE analysis (Coomassie stained). Non-reducing (ox) conditions show the Fab dimer and a prominent 28 kDa band. After reduction (red), a 14 kDa band is observed. (C) Mass spectrometry analysis results, illustrated by the amino acid sequence of L19 IgG Fc domain. A sample after pepsin digest was analyzed by ESI-TOF. Two prominent masses were found: The Fab dimer (calculated: 96061.5, observed: 96061.2 Da) and a fragment (calculated: 27857.1 Da, observed: 27856.3 Da) corresponding to a disulfide connected CH3 domain cleaved at the indicated site (asterisk). Sequence analysis was performed by tryptic in-gel digest of the 28 kDa and 14 kDa band in (B) followed by ESI-Q-TOF. Only sequences of the CH3 domain were observed. Bold: Immunoglobuline domains (CH2 and CH3). Underlined: Sequences found in the 28 kDa and 14 kDa band. Dashed underlined: Sequences only found in the 14 kDa band.