Identification and Affinity Determination of Protein-Antibody and Protein-Aptamer Epitopes by Biosensor-Mass Spectrometry Combination

Abstract

Analytical methods for molecular characterization of diagnostic or therapeutic targets have recently gained high interest. This review summarizes the combination of mass spectrometry and surface plasmon resonance (SPR) biosensor analysis for identification and affinity determination of protein interactions with antibodies and DNA-aptamers. The binding constant (KD) of a protein–antibody complex is first determined by immobilizing an antibody or DNA-aptamer on an SPR chip. A proteolytic peptide mixture is then applied to the chip, and following removal of unbound material by washing, the epitope(s) peptide(s) are eluted and identified by MALDI-MS. The SPR-MS combination was applied to a wide range of affinity pairs. Distinct epitope peptides were identified for the cardiac biomarker myoglobin (MG) both from monoclonal and polyclonal antibodies, and binding constants determined for equine and human MG provided molecular assessment of cross immunoreactivities. Mass spectrometric epitope identifications were obtained for linear, as well as for assembled (“conformational”) antibody epitopes, e.g., for the polypeptide chemokine Interleukin-8. Immobilization using protein G substantially improved surface fixation and antibody stabilities for epitope identification and affinity determination. Moreover, epitopes were successfully determined for polyclonal antibodies from biological material, such as from patient antisera upon enzyme replacement therapy of lysosomal diseases. The SPR-MS combination was also successfully applied to identify linear and assembled epitopes for DNA–aptamer interaction complexes of the tumor diagnostic protein C-Met. In summary, the SPR-MS combination has been established as a powerful molecular tool for identification of protein interaction epitopes.

Anti‐CD30 (Ber‐H2) epitope requires structural elements as shown by mass spectroscopy and dual‐site associated kinetics

“…To the uninitiated, mass spectrometry has a wide‐spanning role in proteomics 18,19 and beyond 20‐22 . There is no shortage of methodologies published 23,24 covering many of the aspects required for the successful use of the technology, from protein digestion to data analysis 25,26 and sequencing 27‐29 .…”

“…To the uninitiated, mass spectrometry has a wide-spanning role in proteomics 18,19 and beyond. [20][21][22] There is no shortage of methodologies published 23,24 covering many of the aspects required for the successful use of the technology, from protein digestion to data analysis 25,26 and sequencing. [27][28][29] For our purposes, our protein of interest was cleaved into smaller peptide sequences, physically separated, and then subjected to electrospray ionization MS.…”

Epitope Determination of DNA and RNA Aptamers as Antibody Alternatives by Affinity-Mass Spectrometry Open New Perspectives for Peptide Biomarkers and Molecular Diagnostics

“…Tryptic digestion was performed with an enzyme-substrate ratio of 1:100. High pressure digestion was performed with a Barocycler 2320EXT instrument (Pressure Biosciences, Boston/USA) [2]. Affinity microcolumns were prepared with NHS/EDC-activated Sepharose microbeads, using 40 µg aptamer.…”

“…Affinity determinations of the aptamer complexes with the C-Met protein were carried out by SPR analysis on self-assembled monolayer (SAM) gold chips, by immobilization of either the aptamers or the protein on the chip surface using standard SAM technology [2]. Binding constants were determined with dilution series of protein and aptamers, respectively.…”

“…We report here the first epitope and affinity determinations illustrated for DNA protein aptamers in comparison to monoclonal and polyclonal antibodies. Epitopes were identified for two DNA aptamers (60 and 64 bases) against C-MET, a cancer diagnostic protein, obtained by SELEX from a random DNA library [2]. Proteolytic affinity-mass spectrometry in combination with SPR biosensor analysis (PROTEX-SPR-MS) was used as the principal tool for epitope determination [3].…”