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

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2008

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Perdivara, Irina
Deterding, Leesa J.
Tomer, Kenneth B.

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Analytical and Bioanalytical Chemistry. 2008, 391(1), pp. 325-336. ISSN 1618-2642. eISSN 1618-2650. Available under: doi: 10.1007/s00216-008-1941-z

Zusammenfassung

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.

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Fachgebiet (DDC)
540 Chemie

Schlagwörter

beta-Amyloid antibody, Plaque specific, De novo sequence determination, Microheterogeneity, CDR variations, LC-MS/MS

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ISO 690PERDIVARA, Irina, Leesa J. DETERDING, Adrian MOISE, Kenneth B. TOMER, Michael PRZYBYLSKI, 2008. Determination of primary structure and microheterogeneity of a beta-amyloid plaque-specific antibody using high-performance LC tandem mass spectrometry. In: Analytical and Bioanalytical Chemistry. 2008, 391(1), pp. 325-336. ISSN 1618-2642. eISSN 1618-2650. Available under: doi: 10.1007/s00216-008-1941-z
BibTex
@article{Perdivara2008Deter-9807,
  year={2008},
  doi={10.1007/s00216-008-1941-z},
  title={Determination of primary structure and microheterogeneity of a beta-amyloid plaque-specific antibody using high-performance LC tandem mass spectrometry},
  number={1},
  volume={391},
  issn={1618-2642},
  journal={Analytical and Bioanalytical Chemistry},
  pages={325--336},
  author={Perdivara, Irina and Deterding, Leesa J. and Moise, Adrian and Tomer, Kenneth B. and Przybylski, Michael}
}
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