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Keywords
- Affinity proteomics (1)
- Antibodies* (1)
- Apheresis therapy (1)
- Assay development (1)
- BLAST (1)
- Collision induced dissociation (1)
- Immunoadsorption (1)
- Immunology* (1)
- Ion mobility (1)
- Ligand -Receptor Interactions* (1)
Identification of proteins from apheresis samples was performed by both SDS-PAGE and 2-D gel separation of eluted proteins from staphylococcal protein A-based immunoadsorption columns (Prosorba®) followed by MS peptide mass fingerprinting and MS/MS peptide sequencing on a MALDI QIT TOF mass spectrometer. MS/MS peptide sequencing was performed in conjunction with a micro reversed phase HPLC configured with an online MALDI plate-spotting device. Apheresis treatment had been performed in three patients with longstanding therapy refractory rheumatoid arthritis. 2-D gels displayed ca. 500 spots representing proteins that were eluted from the Prosorba® columns. From 54 gels, a total of 1256 protein spots had been picked and yielded in the identification of 56 non-redundant proteins without counting isoforms. Proteins from the eluates belong to five major groups comprising (i) immunoglobulins (IgG, IgA, IgM heavy and light chains; about 40% of the spots), (ii) proteins involved in coagulation, (iii) HDL/LDL-associated proteins, (iv) proteins from the complement system, and (v) acute phase proteins. MS analysis showed that the full-length C3 complement protein had been cleaved upon complement activation, presumably on the column, such that the anaphylatoxin C3a was produced and released during therapy. Our results are consistent with clinical observations on both patient responses to therapy and reported adverse events. For the first time, direct molecular information has become available to support mechanistic reasoning for the principle of function of staphylococcal protein A-based immunoadsorption therapy and for the explanation of adverse events. According to our results, removal and/or modulation of immune complexes together with complement activation can be regarded as the major events that are taking place during Prosorba® therapy. In order to avoid complement activation and induction of an inflammatory cascade, we suggest the prevention of C3a anaphylatoxin-related reactions during immunoadsorption therapy.
Intact Transition Epitope Mapping - Targeted High-Energy Rupture of Extracted Epitopes (ITEM-THREE)
(2019)
Epitope mapping, which is the identification of antigenic determinants, is essential for the design of novel antibody-based therapeutics and diagnostic tools. ITEM-THREE is a mass spectrometry-based epitope mapping method that can identify epitopes on antigens upon generating an immune complex in electrospray-compatible solutions by adding an antibody of interest to a mixture of peptides from which at least one holds the antibody's epitope. This mixture is nano-electrosprayed without purification. Identification of the epitope peptide is performed within a mass spectrometer that provides an ion mobility cell sandwiched in-between two collision cells and where this ion manipulation setup is flanked by a quadrupole mass analyzer on one side and a time-of-flight mass analyzer on the other side. In a stepwise fashion, immune-complex ions are separated from unbound peptide ions and dissociated to release epitope peptide ions. Immune complex-released peptide ions are separated from antibody ions and fragmented by collision induced dissociation. Epitope-containing peptide fragment ions are recorded, and mass lists are submitted to unsupervised data base search thereby retrieving both, the amino acid sequence of the epitope peptide and the originating antigen. ITEM-THREE was developed with antiTRIM21 and antiRA33 antibodies for which the epitopes were known, subjecting them to mixtures of synthetic peptides of which one contained the respective epitope. ITEM-THREE was then successfully tested with an enzymatic digest of His-tagged recombinant human β-actin and an antiHis-tag antibody, as well as with an enzymatic digest of recombinant human TNFα and an antiTNFα antibody whose epitope was previously unknown.
We have developed a method to determine apparent activation energies of dissociation for ionized protein–protein complexes in the gas phase using electrospray ionization mass spectrometry following the Rice-Ramsperger-Kassel-Marcus quasi-equilibrium theory. Protein–protein complexes were formed in solution, transferred into the gas phase, and separated from excess free protein by ion mobility filtering. Afterwards, complex disassembly was initiated by collision-induced dissociation with step-wise increasing energies. Relative intensities of ion signals were used to calculate apparent activation energies of dissociation in the gas phase by applying linear free energy relations. The method was developed using streptavidin tetramers. Experimentally determined apparent gas-phase activation energies for dissociation (E#A m0g) of complexes consisting of Fc parts from immunoglobulins (IgG-Fc) and three closely related protein G' variants (IgG-Fc•protein G'e, IgG-Fc•protein G'f, and IgG-Fc•protein G'g) show the same order of stabilities as can be inferred from their in-solution binding constants. Differences in stabilities between the protein–protein complexes correspond to single amino acid residue exchanges in the IgG-binding regions of the protein G' variants.
