The protein primary structure of the recombinant Peptibody were investigated systematically by Liquid Chromatography-Mass Spectrometry (LC-MS). The 15 amino acids of N-terminal were Met-Gln-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Lys-Ser-Gly-Gly and the C-terminal was Lys (K), the same as the theoretical sequence. With more proteases, the whole sequence was detected at the coverage of trypsin 87.5%, chymotrypsin 75.3% and Glu-C 76.7%. The peptide-mapping could be used as an valuable standard to certify the complete expression and primary structure of Peptibody. The pI and MW were 8.93 and 37.415 kDa, within the errors allowed . The binding specificity after production were analyzed using anti-VEGFA and anti-His antibodies.

Peptibody was treated with six proteolytic enzymes (trypsin, chymotrypsin, Asp-N, Glu-C, Lys-C and Lys-N) and digested into amenable peptide fragments for LC-MS analysis. The mass spectrometer used was Q Exactive (Thermo Scientific). For sequencing and peptide-mapping, the data were processed by BiopharmaLynx 1.3 sofware. For molecular weight (MW), the data were calculated through deconvolution by ProMass for XcaliburTM software.