Combined with expression of representative antibodies found in serum, antigen affinity and epitope mapping studies, and Rosetta modeling of antibodyCantigen complexes, we show here that this pipeline can provide completely unique insights on the nature of humoral responses elicited by vaccination through a direct proxy of the serum antibody vaccine response. via the elicitation of serum antibodies, yet more than 100 y after the discovery of antibodies, the molecular composition of the human serum antibody repertoire to an antigen remains unknown. Using high-resolution liquid chromatography tandem MS proteomic Anastrozole analyses of serum antibodies coupled with next-generation sequencing of the V gene repertoire in peripheral B cells, we have delineated the human serum IgG and B-cell receptor repertoires following tetanus toxoid (TT) booster vaccination. We show that the TT+ serum IgG repertoire comprises 100 antibody clonotypes, with three clonotypes accounting for >40% of the response. All 13 recombinant IgGs examined bound to vaccine antigen with challenge; (= day 0 or at = day 56 and beyond. The peripheral blood concentration of TT-specific mBCs remained relatively constant from = 40 d to = 169 d (Fig. S1). The VH repertoires for each donor, Anastrozole encoded by day 7 plasmablasts and by IgD? mBCs collected on both day 7 and 3 mo postboost, were determined by 454 (Roche Diagnostics GmbH) sequencing (70,326 and 157,089 high-quality VH reads for HD1 and HD2, respectively; Table S1) and indexed by their VH KIAA0564 clonotype. The VH clonotype, which represents a cluster of antibodies that likely originate from a single B-cell lineage (27, 28), is defined here as the group of VH sequences that share germ-line V and J segments and also exhibit greater than 90% amino acid identity in the complementarity-determining region (CDR)-H3 (threshold for CDR-H3 amino acid identity determined by analysis of test sets from clustered deep-sequencing data; Fig. S2). We observed that the day 7 TT+ plasmablast samples comprised 922 and 538 VH clonotypes for HD1 and HD2, respectively. Serum Proteomics of the TT-Specific IgG Repertoire. The TT+ serum IgG Anastrozole repertoires at = day 0, = 7 d, = 3 mo, and = 9 mo postboost were analyzed using recently developed LC-MS/MS proteomic methodology (20). Importantly, in F(ab)2 resulting from trypsin digestion of IgG, the presence of a conserved cleavage site (Arg) directly upstream of the CDR-H3 and at the fourth residue of the downstream CH1 constant region (Lys) consistently yields a peptide encompassing the highly informative CDR-H3 and the J region (Fig. S3). Proteolysis of the F(ab)2 with other selective proteases (e.g., GluC/LysC) resulted in peptide identifications of very few additional clonotypes (<8% additional high-confidence identifications of those found in trypsinized sample for HD2 at day 0), the vast majority of which were of low abundance. For peptide identifications, a custom database of the antibody repertoire was built using high-quality V gene sequences from the peripheral B cells in Anastrozole each donor (Table S1), in conjunction with a standard shotgun proteomic pipeline with a high-mass accuracy filter (average mass deviation <1.5 ppm) to minimize false identifications (20). Frequencies of antigen affinity chromatography elution- and flow-throughCderived CDR-H3 peptides mapping to a unique clonotype in the 454 donor-specific sequence database are shown in Fig. 1. The serum IgG clonotype frequency histograms are highly reproducible among technical replicates (20). Open in a separate window Fig. 1. Representative histogram of antibody clonotype frequencies identified proteomically in the F(ab)2 elution and flow-through fractions following TT affinity purification. The histogram shown depicts the 3-mo postboost serum IgG repertoire for HD1. Frequencies shown here were calculated Anastrozole by adding the CDR-H3 spectral counts for all peptides mapping to a single clonotype. Sensitivity and Resolution of CDR-H3 Peptide Quantitation. To determine the dynamic range of detection of serum antibodies and to calibrate the resolution of antibody quantitation, isotopically labeled peptides corresponding to seven TT-specific CDR-H3 sequences observed over a wide range of MS peak intensities in serum samples from donor HD1 and ranging from 15 to 25 residues in length (i.e., largely.
