Sequencing of monoclonal antibody 1D3 (mAb1D3)

De novo sequencing and expression of recombinant 1D3 antibody (R1D3). To achieve maximum coverage for antibody sequencing, 5 µg of the antibody original 1D3 was digested in parallel with five different proteases: trypsin, elastase, chymotrypsin, and Asp-N. For each digestion mixture, peptides were loaded onto a nanoflow C18 HPLC column, and peptides were resolved using an aqueous to organic gradient over the course of 90 minutes. As they eluted from the column, peptides were directly ionized on a Thermo Fisher Orbitrap orbitrap Velos mass spectrometer. In a data-dependent manner, both high-resolution full mass measurements and multiple different tandem mass fragmentation (MS/MS) modalities were collected to give the greatest likelihood of correct sequence interpretation. These include standard collision-induced dissociation (CID), higher-energy dissociation (HCD), and electron transfer dissociation (ETD).
After acquisition, data were transferred to Abterra Bioscience for analysis using their proprietary Valens platform. Briefly, an analysis of bottom-up mass spectra generated by the Vanderbilt University Proteomics facility using multiple enzymes was conducted. The framework sequence was identified by performing a database search of the spectra against the germline immunoglobulin gene sequences.