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 (K_D) 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.

Keywords: DNA aptamers; SPR; affinity determination; chip-MALDI-mass spectrometry; cross-immunoreactivity; epitope structure determination; interleukin-8; monoclonal; myoglobin; polyclonal protein antibodies; proteolytic epitope extraction.

Figure 1

Scheme of the analytical platform for the Chip-SPR–MALDI-MS epitope analyzer. Autosampler unit with SPR chip—MALDI-MS connection and transfer line from SPR valve to MALDI-MS sample target (multiwell plate). Sample injection is performed on the SPR chip containing the immobilized antibody. Peptides injected on the antibody channel provide the SPR sensorgram of the binding epitope(s), and eluted epitope peptide(s) are spotted on the MALDI target plate. K_D determinations of the antibody-bound protein and peptide fragments are performed by injecting a dilution series over the SPR chip.

Figure 2

SPR interaction kinetics of horse heart myoglobin (A) and human myoglobin (B) with polyclonal anti-MG antibody immobilized on protein G [38].

Figure 3

Identification of epitopes of horse heart myoglobin by chip-SPR-MALDI-MS with a monoclonal antibody (A) and a polyclonal antibody (B). Antibodies were immobilized by protein G- covalent fixation as described in Materials and Methods, and tryptic digest mixtures of myoglobin submitted on the chip. Sequence of MALDI spectra for each antibody denote the supernatant (1a), background after final washing (2), and elution with 0.01 M aqueous TFA (3). Epitope peptides identified are illustrated in the myoglobin structure, (146–153) for monoclonal (red) [38], and (79–96) for the polyclonal antibody (yellow).

Figure 4

Elution of α-chymotryptic digest mixture of Adalimumab from immobilized serum (A) and MALDI-MS identification of the epitope peptide (12–29) (B).

Figure 5

Identification of the interleukin-8 epitope to a monoclonal anti-IL8 antibody by proteolytic extraction MALDI-MS, upon elution of the bound epitopes from a microaffinity column (A), and elution from an SPR chip (B). The antibody was immobilized by NHS/EDC coupling as described [53]. Epitope elution provided a discontinuous epitope comprising IL-8 sequences (12–20) and (55–60).

Figure 6

Tertiary structure docking of the αGal (309–322) epitope in the enzyme structure (A) and ESI-mass spectrum of the epitope from the affinity elution fraction after α−Gal tryptic digestion (B). The m/z values of peaks correspond to the charge states of multiply protonated peptide ions [62].

Figure 7

Epitope identification of the complex of the cancer diagnostic protein C-Met with the DNA-aptamer CLN-004 by tryptic epitope extraction from immobilized aptamer on CNBr activated Sepharose affinity column [69]. (A); SPR chip-MALDI-MS upon tryptic epitope extraction from immobilized CLN0004 on an SAM-coated SPR chip (B), MALDI-MS upon epitope extraction between C-Met and CLN0004 aptamer.