Search Results (3)

Precision medicine requests accurate serological inspections to precisely stratify patients for targeted treatment. Intact transition epitope mapping analysis proved surrogate seroconversion of a model organism’s serum when spiked with a monoclonal murine anti-Ovalbumin antibody (mAb) with epitope resolution. Isolation of the IgG fraction from blood serum applied two consecutive protein precipitation steps followed by ultrafiltration and resulted in an ESI-MS analysis-ready IgG preparation. For epitope mapping by epitope extraction, the Ovalbumin antigen was digested with trypsin. After desalting, the peptide mixture was added to the ESI-MS-ready IgG preparation from mAb-spiked serum and the solution was incubated to form an immune complex between the Ovalbumin-derived epitope peptide and the anti-Ovalbumin mAb. Then, the entire mixture of proteins and peptides was directly electrosprayed. Sorting of ions in the mass spectrometer’s gas phase, dissociation of the immune complex ions by collision-induced dissociation, and recording of the epitope peptide ion that had been released from the immune complex proved the presence of the anti-Ovalbumin mAb in serum. Mass determination of the complex-released epitope peptide ion with isotope resolution is highly accurate, guaranteeing high specificity of this novel analysis approach, which is termed Intact Transition Epitope Mapping—Serological Inspections by Epitope EXtraction (ITEM—SIX). Full article

Antibody-based point-of-care diagnostics have become indispensable for modern medicine. In-depth analysis of antibody recognition mechanisms is the key to tailoring the accuracy and precision of test results, which themselves are crucial for targeted and personalized therapy. A rapid and robust method is desired by which binding strengths between antigens and antibodies of concern can be fine-mapped with amino acid residue resolution to examine the assumedly serious effects of single amino acid polymorphisms on insufficiencies of antibody-based detection capabilities of, e.g., life-threatening conditions such as myocardial infarction. The experimental ITEM-FOUR approach makes use of modern mass spectrometry instrumentation to investigate intact immune complexes in the gas phase. ITEM-FOUR together with molecular dynamics simulations, enables the determination of the influences of individually exchanged amino acid residues within a defined epitope on an immune complex’s binding strength. Wild-type and mutated epitope peptides were ranked according to their experimentally determined dissociation enthalpies relative to each other, thereby revealing which single amino acid polymorphism caused weakened, impaired, and even abolished antibody binding. Investigating a diagnostically relevant human cardiac Troponin I epitope for which seven nonsynonymous single nucleotide polymorphisms are known to exist in the human population tackles a medically relevant but hitherto unsolved problem of current antibody-based point-of-care diagnostics. Full article

Electrospray mass spectrometry is applied to determine apparent binding energies and quasi equilibrium dissociation constants of immune complex dissociation reactions in the gas phase. Myoglobin, a natural protein-ligand complex, has been used to develop the procedure which starts from determining mean charge states and normalized and averaged ion intensities. The apparent dissociation constant $K_{D m0g}^{\#}=$ 3.60 × 10⁻¹² for the gas phase heme dissociation process was calculated from the mass spectrometry data and by subsequent extrapolation to room temperature to mimic collision conditions for neutral and resting myoglobin. Similarly, for RNAse S dissociation at room temperature a $K_{D m0g}^{\#}=$ 4.03 × 10⁻¹² was determined. The protocol was tested with two immune complexes consisting of epitope peptides and monoclonal antibodies. For the epitope peptide dissociation reaction of the FLAG peptide from the antiFLAG antibody complex an apparent gas phase dissociation constant $K_{D m0g}^{\#}=$ 4.04 × 10⁻¹² was calculated. Likewise, an apparent $K_{D m0g}^{\#}=$ 4.58 × 10⁻¹² was calculated for the troponin I epitope peptide—antiTroponin I antibody immune complex dissociation. Electrospray mass spectrometry is a rapid method, which requires small sample amounts for either identification of protein-bound ligands or for determination of the apparent gas phase protein-ligand complex binding strengths. Full article