Facile Fabrication and Instant Application of Miniaturized Antibody- Decorated Affinity Columns for Higher-Order Structure and Functional Characterization of TRIM21 Epitope Peptides M. Al-Majdoub, † K. F. M. Opuni, † C. Koy, and M. O. Glocker* Proteome Center Rostock, University Medicine Rostock, Rostock, Germany * S Supporting Information ABSTRACT: Both epitope excision and epitope extraction methods, combined with mass spectrometry, generate precise informations on binding surfaces of full-length proteins, identifying sequential (linear) or assembled (conformational) epitopes, respectively. Here, we describe the one-step fabrication and application of affinity columns using reversibly immobilized antibodies with highest flexibility with respect to antibody sources and lowest sample amount requirements (fmol range). Depending on the antibody source, we made use of protein G- or protein A-coated resins as support materials. These materials are packed in pipet tips and in combination with a programmable multichannel pipet form a highly efficient epitope mapping system. In addition to epitope identification, the influence of epitope structure modifications on antibody binding specificities could be studied in detail with synthetic peptides. Elution of epitope peptides was optimized such that mass spectrometric analysis was feasible after a single desalting step. Epitope peptides were identified by accurate molecular mass determinations or by partial amino acid sequence analysis. In addition, charge state comparison or ion mobility analysis of eluted epitope peptides enabled investigation of higher-order structures. The epitope peptide of the TRIM21 (TRIM: tripartite motif) autoantigen that is recognized by a polyclonal antibody was determined as assembling an “L-E-Q-L” motif on an α-helix. Secondary structure determination by circular dichroism spectroscopy and structure modeling are in accordance with the mass spectrometric results and the antigenic behavior of the 17-mer epitope peptide variants from the full-length autoantigen. M ass spectrometric epitope mapping approaches have been developed as powerful tools to identify molecular details of antigen-antibody interactions. 1,2 Various related approaches used for epitope mapping include HPLC, 3 chemical modification 4,5 of surface exposed residues on the antigen and differential analysis upon shielding by antibody complexation, analysis of hydrogen/deuterium exchange 6 differences on the antigen with and without antibody binding, Western blot analysis with chemically produced antigen fragments 7 or with fusion proteins 8 that contain partial sequences of the antigen, and affinity chromatography-related approaches. 9-11 Mass spectrometric epitope mapping analyses are usually divided into epitope excision 2,12 and epitope extraction. 1,13 The interaction structure analysis principle has been extended to map antibody paratopes 11 and to the analysis of carbohydrate binding sites on lectins, 14,15 respectively. Mass spectrometric epitope excision or epitope extraction experiments are mostly performed with chemically immobilized antibodies on a support material, thereby generating an affinity column, and successful applications have been reported, for example, by tresyl-activated sepharose magnetic nitrocellulose beads 2 or cyanogen bromide-activated sepharose beads, 4,12,16-18 to which the antibody was attached. Advantages of this experimental setting are that the affinity column can be reused after regeneration procedures, although not indefinitely. Miniatur- ization lead to the production of commercial affinity systems embedded in pipet tips. 19 In contrast, only a few examples have been reported in which neither the antibody nor the antigen were immobilized for epitope mapping. 1,6,9,20 Advantages of this procedure are the avoidance of unwanted chemical modifications of the antibody during immobilization (since poorly controlled) and main- tenance of “nondenaturing” conditions during all steps of the experiment. Reversible noncovalent antibody immobilization using protein A- or protein G-coated columns have found a vast number of applications in immunotechnology, mostly for affinity chromatography of antibodies from complex biological samples. 21,22 The concept of noncovalent immobilization of an antibody on agarose decorated with a protein G/A mixture for mapping the epitopes on small peptides, such as melittin and glucagon-like peptide-1 7-37 (GLP-1 7-37), respectively, has been reported in a single instance. 23 Epitope mapping of autoantigens has become important for designing so-called “next generation chip arrays”. 24 Disease- specific epitope peptides need to be identified and characterized Received: August 6, 2013 Accepted: October 4, 2013 Article pubs.acs.org/ac © XXXX American Chemical Society A dx.doi.org/10.1021/ac402559m | Anal. Chem. XXXX, XXX, XXX-XXX