Greg Ippolito for reading the manuscript, Prof. chromatography elution, constituting the serum polyclonal response. After booster immunization in a rabbit, we find that this antigen-specific serum immune response is usually oligoclonal, comprising antibodies encoding 34 different CDRH3s that group into 30 unique antibody VH clonotypes. Of these 34 CDRH3s, 12 account for 60% of the antigen-specific CDRH3 peptide mass spectral counts. For comparison, antibodies with 18 different CDRH3s (12 clonotypes) were represented in the antigen-specific IgG portion from an unimmunized rabbit that fortuitously displayed a moderate titer for BSA. Proteomically recognized antibodies were synthesized and shown to display subnanomolar affinities. The ability to deconvolute the polyclonal serum response is likely to be of important importance for analyzing antibody responses after vaccination and for more completely understanding adaptive immune responses in health and disease. Keywords: antibody proteomics, antibody repertoire, serum immunoprofiling, B-cell response, humoral response The first Nobel Prize in Medicine was awarded to Emil von Behring, who in collaboration with Kitasato Shibasaburo and Paul Ehrlich discovered serum antitoxins (1, 2). Amazingly, after more than 100 y of intense research in immunology, little is known about the clonality, relative concentrations, and binding properties of the monoclonal antibodies that constitute the antigen-specific Ig pool in serum. At constant state, circulating antibodies are produced by terminally differentiated B lymphocytes (plasma cells) within the bone marrow, and thus cannot be utilized in living individuals (3). Although recent single B-cell cloning methods (4, 5) have led to the identification of peripheral antigen-specific B memory and/or antibody-secreting cells (plasmablasts), it is generally unknown whether the Igs encoded by peripheral blood B Chlorpheniramine maleate cells correspond to the antibodies present in circulation and especially whether they are present at physiologically relevant levels (i.e., at serum concentrations above KD corresponding to >1 g/mL for an average affinity of individual antibodies of 5 nM). The proteomic deconvolution of serum Igs presents two major technical difficulties: first, antibody genes in antigen stimulated B-lymphocytes are not just encoded in the germline but are extensively diversified by somatic recombination, revision, and/or mutation. Therefore, the sequence database required for the interpretation of mass spectra is not available a priori (6, 7) and is completely different for each individual. Second, the antigen-specific antibody pool comprises a wide variety of Igs that display very high levels of amino acid identity within the framework regions. As a result, RCAN1 standard methods for proteomic analysis by MS are confounded by this exceptionally high rate of identical sequence shared among Ig-derived peptides, which greatly complicates the task of confidently identifying individual variable (V) genes through peptide mapping. Developments in sequencing and MS technologies have shown some success against these difficulties. MS-based de novo sequencing methods have been utilized for the identification of purified monoclonal antibodies (8). More recently the identification of a limited subset of antigen-specific antibodies in serum after very stringent enrichment to reduce the complexity of the antigen-specific polyclonal antibody pool to a limited set of Igs from humans and animals was reported (9C12). However, because of the inherent troubles associated with the proteomic analysis of complex mixtures of antibodies, these studies had focused on the identification of only a small subset of the Chlorpheniramine maleate antigen-specific serum IgGs present in a portion isolated after stringent affinity chromatography. In contrast, complete understanding of how B-cell differentiation ultimately designs humoral immunity requires addressing the more Chlorpheniramine maleate difficult problem of how to deconvolute the entire repertoire of antigen-specific antibodies in serum or in other secretions. Here we describe the proteomic deconvolution of the serum-derived antigen-specific polyclonal antibody pool by combining NextGen sequencing of the immunoglobulin heavy chain variable region (VH gene) repertoire with liquid chromatographyChigh-resolution tandem mass spectrometry (LC-MS/MS) (Fig. 1). Proteomic identifications of unique VH-derived peptides (overwhelmingly from your CDR3 region of the VH sequences) were used to determine the VH repertoire of circulating antigen-specific antibodies, and recognized VH genes were shown to encode antibodies with subnanomolar antigen affinity. Open in a separate windows Fig. 1. Schematic of the workflow for serum Ig deconvolution. (= 4729 VH genes, blue bars), peripheral B cells (= 2788 VH genes, reddish bars), or from antibody proteins recognized by proteomic analysis of the serum affinity purified IgGs (= 334, green bars) for the CCH rabbit. To generate antibodies, proteomically recognized VH sequences must be paired with the VL genes. We therefore synthesized VH domain name DNAs and used these to construct phage displayed scFv libraries comprising the VL cDNA repertoire. Functional scFv antibodies were isolated after two to three rounds of phage panning. Putative pairings of VH and VL genes were then expressed recombinantly as full-size IgGs in mammalian cells and isolated to characterize.