Conférence de chimie
Titre complet
New Perspectives for Proteomics, Biomedical and Biomolecular Epitope, Determination by Combination of Affinity Tools and Mass Spectrometry
Cette conférence sera prononcée (en anglais) par le Professeur Michael Przybylski du Steinbeis Center for Biopolymer Analysis and Biomedical Mass Spectrometry, University of Konstanz.
Hôte : Joelle Pelletier
Résumé:
Bioaffinity-based technologies such as ELISA, Western Blot and biosensor determination are long established in the analysis of biomolecular interactions, but their combination with mass spectrometry (MS) is only beginning to be explored. Bioaffinity and MS technologies are recently emerging as powerful “hybrid” tools for detection, chemical structure determination and quantification of biomolecular interactions, particularly recognition epitopes. New developments of MS for the characterization of biopolymer interactions will be reviewed by combination with biochemical affinity techniques; affinity-separation, affinity determination and quantification; identification of antigen epitopes and antibody paratopes; clinical applications of affinity- protomics. These technologies are currently gaining high interest in many application areas such as pharmacology, clinical diagnostics and the development of antibody-based drugs.
Bioaffinity analysis using biosensors such as surface plasmon resonance (SPR) has become an established technique for the detection and quantification of biomolecular interactions. However, a principal limitation of biosensors is their lack of providing chemical structure information of affinity-bound ligands. Proteolytic excision/extraction (Protex-MS), hydrogen-deuterium exchange (HDX-MS) of peptide backbone hydrogens, and Fast- Photochemical Oxidation (FPOP) are major techniques for mass spectrometry based elucidation of protein- ligand interactions, but none of these tools alone provide quantitative affinity data.
We have developed an online SPR- biosensor-MS combination with electrospray ionization mass spectrometry that enables the simultaneous affinity isolation, chemical structure determination and affinity quantification of protein and peptide epitopes [Patent pending; 2016]. Key tool of the SPR- MS combination is an integrated, automated interface that provides sample concentration and in-situ desalting for MS analysis of the ligand eluate [1]. ESI-MS instruments from all major manufacturers and a wide range of biosensors (in addition to SPR) can be coupled. Recent applications of the online SPR- MS show broad bioanalytical potential and high performance for interaction studies and epitope characterization from biological material, as diverse as antigen-antibody and lectin- carbohydrate complexes; affinity binding constants (KD) determinations are well feasible from milli- to nanomolar ranges [2, 3]. First applications to the direct analysis of biological samples, such as cell lysate and tissue homogenate will be discussed.
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- Przybylski, M. et al., (2016) Anal. Bioanal. Chem. In press.
