Molecular epitope determination of aptamer complexes of the multidomain protein CMet by affinity-mass spectrometry

Michael Przybylski, Steinbeis Centre for Biopolymer Analysis and Biomedical Mass Spectrometry, Rüsselsheim am Main, Germany (michael.przybylski@stw.de)
Loredana Lupu, Steinbeis Centre For Biopolymer Analysis And Biomedical Mass Spectrometry, Rüsselsheim Am Main, Germany
Pascal Wiegand, Steinbeis Centre For Biopolymer Analysis And Biomedical Mass Spectrometry, Rüsselsheim Am Main, Germany
Nico Hüttmann, Steinbeis Centre For Biopolymer Analysis And Biomedical Mass Spectrometry, Rüsselsheim Am Main, Germany
Stephan Rawer, Steinbeis Centre For Biopolymer Analysis And Biomedical Mass Spectrometry, Rüsselsheim Am Main, Germany
Wolfgang Kleinekofort, Steinbeis Centre For Biopolymer Analysis And Biomedical Mass Spectrometry, Rüsselsheim Am Main, Germany
Friedemann Völklein, Dept. Engineering, Rhein Main University, Rüsselsheim Am Main, Germany
Günes Barka, Sunchrom Gmbh, Friedrichsdorf, Germany
Alexander Lazarev, Pressure Biosciences Inc., Boston, Usa
Maxim Berezovski, Dept. Of Biochemistry, University Of Ottawa, Ottawa, Canada

We present here a new approach for  identification of biomarkers of pathophysiological target proteins, and alternative to pathophysiological antibodies, by molecular epitope identification of DNA-aptamers. C-Met protein has been recognized as a biomarker in cancer diagnosis by transmitting intracellular signals due to a unique multi-substrate docking site, and has been found to be aberrantly activated leading to tumorigenesis. C-Met aptamers have been recently considered a valuable tool for detection of cancer biomarkers.  Aptamers are single-stranded DNA or RNA oligonucleotides that are readily produced and show stabilities and affinities comparable to monoclonal antibodies. A molecular interaction study of human C-Met protein expressed in kidney cells was performed with two DNA aptamers of 60 and 64 bases (CLN003 and CLN004), prepared and isolated using the SELEX procedure. Specific epitope peptides of the C-Met aptamers were identified by proteolytic affinity- mass spectrometry in a new combination with an SPR biosensor (PROTEX-SPR-MS), using high pressure proteolysis as an efficient tool for proteolytic digestion- epitope extraction of native and denatured C-Met. High affinities with binding constants K_D of 80 - 510 nM were determined for aptamer- C-Met complexes, with a two-step binding pathway suggested by kinetic analyses. A single linear epitope peptide, C-Met(381-393) (NSSGCEARRDEYR) was identified for CLN-0004, while the CLN-0003 aptamer revealed an assembled epitope comprised of two specific peptide sequences^[1]. Epitope specificities and affinities were ascertained by characterization of synthetic epitope peptides. The high specificities  and affinities of C-Met aptamers in this study suggest high potential for molecular diagnostics. Moreover, on the basis of the epitope identifications, aptamers with high affinity can be developed for therapeutic intervention such as neutralization of pathophysiological antibodies^[2] against therapeutic proteins.

References

[1] Lupu, L. et al. 2018, Proc. Int. Mass Spec. Conf., 241.

[2] Kukacka Z., et al. 2018, ChemMedChem. 13: 909-915.

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Abstract Reference & Short Personal Biography of Presenting Author

Michael Przybylski studied chemistry and completed his PhD dissertation at the University of Mainz. He spent two years as a Visiting Scientist at the National Cancer Institute, NIH/USA, and was appointed in 1989 to the Chair of Analytical Chemistry at the University of Konstanz, where he was the Director of the Laboratory of Analytical Chemistry and Biopolymer Structure Analysis. Michael Przybylski is currently Director of the Steinbeis Centre for Biopolymer Analysis and Biomedical Mass Spectrometry, located for 8 years at the University of Konstanz, and located in Rüsselsheim am Main since 2016. Since 2 years the Laboratory is located in Rüsselsheim am Main; it is associated with the Rhein Main University Rüsselsheim, Dept. of Engineering Sciences. The laboratory’s current research is focused on the development of biosensor and mass spectrometry technologies for elucidation of antibody epitopes; applications of mass spectrometry to structure elucidation and and pathophysiological modifications of lysosomal proteins;  structure analysis of therapeutic antibodies; development of aptamers as surrogate antibodies; affinity-mass spectrometry of biopolymer interaction epitopes. His laboratory has made numerous new developments and applications of biopolymer mass spectrometry methods, and has invented proteolytic‐ affinity mass spectrometry for the elucidation of protein‐ligand interaction structures, and peptide/protein epitopes. He has been member of Scientific Committees of several International Conferences, Editor and Editorial Board member of several International Journals, and has been President of the German Society for Mass Spectrometry. He has published over 400 scientific publications in peer reviewed International Journals; 6 monographs/reviews; ca. 25 patents; and given ca. 150 invited lectures.