TITLE

Determination of primary structure and microheterogeneity of a β-amyloid plaque-specific antibody using high-performance LC–tandem mass spectrometry

AUTHOR(S)
Perdivara, Irina; Deterding, Leesa; Moise, Adrian; Tomer, Kenneth B.; Przybylski, Michael
PUB. DATE
May 2008
SOURCE
Analytical & Bioanalytical Chemistry;May2008, Vol. 391 Issue 1, p325
SOURCE TYPE
Academic Journal
DOC. TYPE
Article
ABSTRACT
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-β-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 VL 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 not available: see fulltext.]
ACCESSION #
31722419

 

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