Each antibody sample was digested with six different enzymes, Trypsin, Lys C, Chymotrypsin, Pepsin, Asp N, and Elastase (Promega, WI, US). Tandem mass spectrometry (MS/MS) data were collected with an Orbitrap Q Exactive (Thermo Fisher, CA, US) for each digest.
Novor software3 was used to de novo sequence each peptide. The protein sequencing was assembled with the REmAb® sequencing platform (Rapid Novor, Kitchener, ON, Canada) (Figure 1).
After sequencing of the first pair of heavy and light chains for each antibody, the software then sequenced a second light chain from the data.
A second light chain was reported as found if it had significant sequence coverage based on liquid chromatography (LC)-MS/MS data and was sufficiently different from the first one.
Figure 1. General workflow of REmAb® includes (1) multiple enzyme digests, (2) mass spec, (3) de novo peptide sequencing, and (4) sequence assembly.