Automated de novo protein sequencing of monoclonal antibodies

Abstract

De novo protein sequencing of monoclonal antibodies is required when the cDNA or the original cell line is not available, or when characterization of posttranslational modifications is needed to verify antibody integrity and effectiveness. We demonstrate that Comparative Shotgun Protein Sequencing (CSPS) based on tandem mass spectrometry can reduce the time required to sequence an antibody to 72 hours, a dramatic reduction as compared to the classical technique of Edman degradation. We therefore argue that CSPS has the potential to be a disruptive technology for all protein sequencing applications.

Figure 1

Protein contigs resulting from Shotgun Protein Sequencing of aBTLA tandem mass spectra. Left: Protein contig resulting from 24 spectra from the aBTLA heavy chain. Each spectrum is shown superimposed with a sequence of arrows indicating its sequence of recovered masses; modified variants of the consensus sequence are indicated by red arrows (6 different modifications on 7 spectra). Right: Protein contig illustrating the variety of discovered modifications on or next to Cys-146 (aBTLA heavy chain); modified residues are indicated with red labels next to the corresponding edge: C+57 (the expected mass, spectra 1,7, not highlighted), C+71 (acrylamide adduct, spectra 2,3,8,11), C-34 (DeHydroAlanine (DHA), spectra 4,5), DHA+16 (oxidation, spectrum 6), M+16 (oxidation, spectra 1,3), either C+209 or DHA+243 (spectrum 9), either C+223 or DHA+257 (spectrum 10).

Figure 2

Comparative Protein Sequencing. The heavy chain contigs matched to two different proteins (gi|148540420 and gi|148686583) homologous to different regions of the aBTLA heavy chain – 9 SPS-contigs matched gi|148540420, 47 SPS-contigs matched gi|148686583 and 8 contigs matched both (see Figure 2). The protein regions matched by the latter were confirmed by a corroborating CLUSTALW alignment of gi|148540420/gi|148686583. a): Homology-derived order of 36 aBTLA heavy chain protein contigs. Each protein contig is represented as a colored dash on the circumference; the color gradient and black arrow at the top indicate the arbitrary contig order (since the contig order is unknown beforehand). Colored arrows indicate the contiguous contig order induced by the homologous proteins and the dashed colored arrows indicate protein contigs in the correct order but separated by sequence gaps. The recovered contig order is indicated by indices next to each contig; negative indices –j indicate that the j-th contig resulted in a reversed sequence (i.e. inferred from a sequence of y-ions rather than b-ions). b): Linear rendering of the homology-induced contig order illustrated in part a). c): The complete aBTLA heavy chain sequence recovered by our approach; highlighted sections were covered by protein contigs (95% coverage) and the missing amino acids were obtained from the homologous protein sequences.

Figure A

Center: Structure of a typical immunoglobulin (antibody) protein. Two identical heavy chains and two identical light chains are connected by disulfide linkages. The antigen-binding site is composed of the variable regions of the heavy and light chains, whereas the effector site of the antibody is determined by the amino acid sequence of the heavy chain constant region. Bottom: Rearrangement of the light chain genes during B lymphocyte differentiation. While the developing B cell is still maturing in the bone marrow, one of the 300 or more V gene segments combines with one of the 5 J gene segments and moves closer to the constant (C) gene segment. Top: Rearrangement of the heavy chain genes. A heavy chain gene contains three segments (V, D, and J) that come together to form the variable region, as well as a constant region. Reproduced with permission from Sinauer Associate Inc. In vertebrates, antibodies are composed of a pair of heavy chains (HC) (~52-70 KDa each) and light chains (LC) (~25 KDa each) which are linked together via disulphide bonds (Fig.1). This staggering diversity is facilitated during the early phase of B lymphocyte development by integration of the immunoglobulin variable (v), diversity (d) and joining (j) gene segments and is primarily manifested in the hyper-variable regions of the heavy and light chains which constitute the complementarity determining regions (CDRs). The CDRs are mounted on a scaffold of framework residues that vary to a lesser extent among antibodies. The remainder of the antibody framework is termed the constant region and follows a more conserved sequence, although variation may also occur here due to mutations at the genetic level or post translational modifications at the protein level.