An integrated top-down and bottom-up proteomic approach to characterize the antigen-binding fragment of antibodies.

Abstract

We have previously shown that different individuals exposed to the same antigen produce antibodies with identical mutations in their complementarity determining regions (CDR), suggesting that CDR tryptic peptides can serve as biomarkers for disease diagnosis and prognosis. Complete Fabs derived from disease specific antibodies have even higher potential; they could potentially be used for disease treatment and are required to identify the antigens toward which the antibodies are directed. However, complete Fab sequence characterization via LC-MS analysis of tryptic peptides (i.e. bottom-up) has proven to be impractical for mixtures of antibodies. To tackle this challenge, we have developed an integrated bottom-up and top-down MS approach, employing 2D chromatography coupled with Fourier transform mass spectrometry (FTMS), and applied this approach for full characterization of the variable parts of two pharmaceutical monoclonal antibodies with sensitivity comparable to the bottom-up standard. These efforts represent an essential step toward the identification of disease specific antibodies in patient samples with potentially significant clinical impact.

DOI: 10.1002/pmic.201300366

Cite this paper

@article{Dekker2014AnIT, title={An integrated top-down and bottom-up proteomic approach to characterize the antigen-binding fragment of antibodies.}, author={Lennard J. Dekker and Si Wu and Martijn M. VanDuijn and Nikolai Toli{\'c} and Christoph Stingl and Rui Zhao and Theo M. Luider and Ljiljana Pasa-Tolic}, journal={Proteomics}, year={2014}, volume={14 10}, pages={1239-48} }