Do-it-yourself de novo antibody sequencing workflow that achieves complete accuracy of the variable regions

Abstract

Antibodies are widely used as research tools or therapeutic agents. Knowing the sequences of the variable regions of an antibody--both the heavy chain and the light chain--is a prerequisite for the production of recombinant antibodies. Mass spectrometry-based de novo sequencing is a frequently used, and sometimes the only approach to gaining this information. Here, we describe a workflow that enables accurate sequence determination of monoclonal antibodies based on mass spectrometry data and freely available software tools. This workflow, which we developed using a homemade anti-FLAG monoclonal antibody as a reference sample, achieved 100% accuracy of the variable regions with clear distinction between leucine (L) and isoleucine (I). Using this workflow, we successfully decoded a monoclonal anti-HA antibody, for which we had no prior knowledge of its sequence. Based on the de novo sequencing result, we generated a recombinant anti-HA antibody, and demonstrated that it has the same specificity, sensitivity, and affinity as the commercial antibody. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/630060v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@180c502org.highwire.dtl.DTLVardef@1064399org.highwire.dtl.DTLVardef@4005d3org.highwire.dtl.DTLVardef@13e65ed_HPS_FORMAT_FIGEXP M_FIG C_FIG