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- Antibody (2) (entfernen)
Organisationseinheit der BAM
Despite their immense and rapidly increasing
importance as analytical tools or therapeutic drugs, the detailed
structural features of particular monoclonal antibodies are
widely unknown. Here, an antibody already in use for diagnostic
purposes and for molecular dosimetry studies in cancer therapy
with very high affinity and specificity for cisplatin-induced DNA
modifications was studied extensively. The molecular structure
and modifications as well as the antigen specificity were
investigated mainly by mass spectrometry. Using nano electrospray
ionization mass spectrometry, it was possible to characterize the antibody in its native state. Tandem-MS experiments not
only revealed specific fragments but also gave information on the molecular structure. The detailed primary structure was further
elucidated by proteolytic treatment with a selection of enzymes and high resolution tandem-MS. The data were validated by
comparison with known antibody sequences. Then, the complex glycan structures bound to the antibody were characterized in
all detail. The Fc-bound oligosaccharides were released enzymatically and studied by matrix-assisted laser desorption/ionization
mass spectrometry. Overall 16 different major glycan structures were identified. The binding specificity of the antibody was
investigated by applying synthetic single and double stranded DNA oligomers harboring distinct Pt adducts. The antibody−
antigen complexes were analyzed by mass spectrometry under native conditions. The stability of the complex with double
stranded DNA was also investigated.
Quantitative analysis of complex proteins is a challenging task in modern bioanalytical chemistry. Commonly available isotope labels are still suffering from limitations and drawbacks, whereas new metal labels open numerous possibilities in mass spectrometric analyses. In this work, we have developed a newmetal labeling strategy to tag glycan structures of proteins, more particularly antibodies. The oligosaccharide glycans were selectively trimmed to the last N-acetylglucosamine to which an artificial azide containing galactose residue was bound. This azide can be used for subsequent cycloaddition of an alkyne. Therefore, we developed a lanthanide-containing macrocyclic reagent to selectively connect to this azido galactose. In summary, the glycan structures of an antibody can be labeled with a metal functionality using this approach. Furthermore, the functionality of the antibodies can be fully maintained by labeling the Fc glycans instead of using labeling reagents that target amino or thiol groups. This approach enables the possibility of using elemental, besides molecular mass spectrometry, for quantitative analyses or imaging experiments of antibodies in complex biological samples.