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Intact Transition Epitope Mapping - Targeted High-Energy Rupture of Extracted Epitopes (ITEM-THREE)

  • 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.

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Metadaten
Document Type:Article
Language:English
Parent Title (English):Molecular & Cellular Proteomics
Volume:18
Issue:8
First Page:1543
Last Page:1555
ISSN:1535-9476
DOI:https://doi.org/10.1074/mcp.RA119.001429
Pubmed Id:http://www.ncbi.nlm.nih.gov/pubmed?term=31147491
Publisher:American Society for Biochemistry and Molecular Biology
Date of first publication:2019/05/30
Tag:Affinity proteomics; Antibodies*; Assay development; BLAST; Immunology*; Ligand -Receptor Interactions*; Mass spectrometry; Non-covalent interaction MS*; Protein complex analysis; Targeted mass spectrometry; Therapeutic antibodies*; data base search; epitope mapping; peptide sequencing
Departments, institutes and facilities:Fachbereich Angewandte Naturwissenschaften
Institut für funktionale Gen-Analytik (IfGA)
Dewey Decimal Classification (DDC):5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Entry in this database:2019/08/06