Primary sequence determination of a monoclonal antibody against α-synuclein using a novel mass spectrometry-based approach

A novel mass spectrometry-based workflow for de novo sequencing of a full-length monoclonal antibody (LB509) against α-synuclein is presented. This approach combines chemical modification of cysteine residues, multiple enzymatic digestion, stable isotope labeled amino acids in cell culture (SILAC), liquid chromatography coupled tandem mass spectrometry (LC/MS/MS) analysis including collision-induced dissociation (CID), higher energy collision-induced dissociation (HCD), and de novo sequencing software for data interpretation. The introduction of aminoethyl-8 (A-8) reagent which renders cysteine residues in the antibody to become substrates for trypsin digestion significantly increases the sequence coverage of the antibody, especially in the complementarity determining regions (CDRs). Incorporation of SILAC helps to distinguish minor sequence variations between original and deduced sequences. Methods in obtaining detailed sequence characterization are described, highlighting the advantage of using multiple fragmentation schemes, CID in ion trap and HCD in C-trap. Complete primary sequence for LB509 has been construed and the antibody generated exhibits specific binding to α-synuclein. The development and implementation of this technology enables an alternative approach for generating therapeutic candidate and reagent antibodies.

Use of visualization tool to aid identification of CDR-containing peptides after A-8 modification and trypsin digestion.Figure optionsDownload high-quality image (83 K)Download as PowerPoint slideHighlights
► A novel mass spectrometry-based approach for de novo sequencing monoclonal antibody.
► Incorporated modification of Cys, SILAC, LC/MS/MS, and de novo software.
► Complete primary sequence for antibody LB509 was construed.
► Reverse-engineered antibody exhibited specific binding to α-synuclein.