Determination of primary structure and microheterogeneity of a beta-amyloid plaque-specific antibody using high-performance LC-tandem mass spectrometry.
Using the bottom-up approach and liquid chromatography (LC) in combination with mass spectrometry, the primary structure and sequence microheterogeneity of a plaque-specific anti-beta-amyloid (1-17) monoclonal antibody (clone 6E10) was characterized. This study describes the extent of structural information directly attainable by a high-performance LC-tandem mass spectrometric method in combination with both protein database searching and de novo sequence determination. Using trypsin and chymotrypsin for enzymatic digestion, 95% sequence coverage of the light chain and 82% sequence coverage of the heavy chain of the 6E10 antibody were obtained. The primary structure determination of a large number of peptides from the antibody variable regions was obtained through de novo interpretation of the data. In addition, N-terminal truncations of the heavy chain were identified as well as low levels of pyroglutamic acid formation. Surprisingly, pronounced sequence microheterogeneities were determined for the CDR 2 region of the light chain, indicating that changes at the protein level derived from somatic hypermutation of the Ig V(L) genes in mature B-cells might contribute to unexpected structural diversity. Furthermore, the major glycoforms at the conserved heavy chain N-glycosylation site, Asn-292, were determined to be core-fucosylated, biantennary, complex-type structures containing zero to two galactose residues. [figure: see text]
Perdivara
Irina
I
Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry, University of Konstanz, 78457, Konstanz, Germany.
Deterding
Leesa
L
Moise
Adrian
A
Tomer
Kenneth B
KB
Przybylski
Michael
M
D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D000918:Antibody Specificity; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006031:Glycosylation; D007135:Immunoglobulin Variable Region / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D058225:Plaque, Amyloid / Q000276:immunology*; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D053719:Tandem Mass Spectrometry / Q000379:methods
D016229:Amyloid beta-Peptides; D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; C078639:amyloid beta-protein (1-38)