Proteomic identification of antibodies

Methods and compositions for identification of candidate antigen-specific variable regions as well as generation of antibodies or antigen-binding fragments that could have desired antigen specificity are provided. For example, in certain aspects, methods for determining amino acid sequences of serum antibody CDR3 and abundancy levels are described. In some aspects, methods for determining nucleic acid sequences of antibody variable region sequences and the frequency thereof in biological samples are provided. Furthermore, the invention provides methods for identification and generation of antibodies or antigen-binding fragments that comprise highly-represented CDR domains.Board of Regents, University of Texas Syste

High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire.

Each B-cell receptor consists of a pair of heavy and light chains. High-throughput sequencing can identify large numbers of heavy- and light-chain variable regions (VH and VL) in a given B-cell repertoire, but information about endogenous pairing of heavy and light chains is lost after bulk lysis of B-cell populations. Here we describe a way to retain this pairing information. In our approach, single B cells (>5 × 104 capacity per experiment) are deposited in a high-density microwell plate (125 pl/well) and lysed in situ. mRNA is then captured on magnetic beads, reverse transcribed and amplified by emulsion VH:VL linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of VH:VL pairs identified by this approach and used the method to sequence the repertoire of three human cell subsets-peripheral blood IgG+ B cells, peripheral plasmablasts isolated after tetanus toxoid immunization and memory B cells isolated after seasonal influenza vaccinatio

Proteomic identification of antibodies

Methods and compositions for identification of candidate antigen-specific variable regions as well as generation of antibodies or antigen-binding fragments that could have desired antigen specificity are provided. For example, in certain aspects, methods for determining amino acid sequences of serum antibody CDR3 and abundancy levels are described. In some aspects, methods for determining nucleic acid sequences of antibody variable region sequences and the frequency thereof in biological samples are provided. Furthermore, the invention provides methods for identification and generation of antibodies or antigen-binding fragments that comprise highly-represented CDR domains.Board of Regents, University of Texas Syste

Selective 351 nm Photodissociation of Cysteine-Containing Peptides for Discrimination of Antigen-Binding Regions of IgG Fragments in Bottom-Up Liquid Chromatography–Tandem Mass Spectrometry Workflows

Despite tremendous inroads in the development of more sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) strategies for mass spectrometry-based proteomics, there remains a significant need for enhancing the selectivity of MS/MS-based workflows for streamlined analysis of complex biological mixtures. Here, a novel LC–MS/MS platform based on 351 nm ultraviolet photodissociation (UVPD) is presented for the selective analysis of cysteine–peptide subsets in complex protein digests. Cysteine-selective UVPD is mediated through the site-specific conjugation of reduced cysteine residues with a 351 nm active chromogenic Alexa Fluor 350 (AF350) maleimide tag. Only peptides containing the AF350 chromophore undergo photodissociation into extensive arrays of <i>b</i>- and <i>y</i>-type fragment ions, thus providing a facile means for differentiating cysteine–peptide targets from convoluting peptide backgrounds. With the use of this approach in addition to strategic proteolysis, the selective analysis of diagnostic heavy-chain complementarity determining regions (CDRs) of single-chain antibody (scAb) fragments is demonstrated

ASAP - A Webserver for Immunoglobulin-Sequencing Analysis Pipeline

Reproducible and robust data on antibody repertoires are invaluable for basic and applied immunology. Next-generation sequencing (NGS) of antibody variable regions has emerged as a powerful tool in systems immunology, providing quantitative molecular information on antibody polyclonal composition. However, major computational challenges exist when analyzing antibody sequences, from error handling to hypermutation profiles and clonal expansion analyses. In this work, we developed the ASAP (A webserver for Immunoglobulin-Seq Analysis Pipeline) webserver (https://asap.tau.ac.il). The input to ASAP is a paired-end sequence dataset from one or more replicates, with or without unique molecular identifiers. These datasets can be derived from NGS of human or murine antibody variable regions. ASAP first filters and annotates the sequence reads using public or user-provided germline sequence information. The ASAP webserver next performs various calculations, including somatic hypermutation level, CDR3 lengths, V(D)J family assignments, and V(D)J combination distribution. These analyses are repeated for each replicate. ASAP provides additional information by analyzing the commonalities and differences between the repeats (“joint” analysis). For example, ASAP examines the shared variable regions and their frequency in each replicate to determine which sequences are less likely to be a result of a sample preparation derived and/or sequencing errors. Moreover, ASAP clusters the data to clones and reports the identity and prevalence of top ranking clones (clonal expansion analysis). ASAP further provides the distribution of synonymous and non-synonymous mutations within the V genes somatic hypermutations. Finally, ASAP provides means to process the data for proteomic analysis of serum/secreted antibodies by generating a variable region database for liquid chromatography high resolution tandem mass spectrometry (LC-MS/MS) interpretation. ASAP is user-friendly, free, and open to all users, with no login requirement. ASAP is applicable for researchers interested in basic questions related to B cell development and differentiation, as well as applied researchers who are interested in vaccine development and monoclonal antibody engineering. By virtue of its user-friendliness, ASAP opens the antibody analysis field to non-expert users who seek to boost their research with immune repertoire analysis

PASA: Proteomic analysis of serum antibodies web server.

MotivationA comprehensive characterization of the humoral response towards a specific antigen requires quantification of the B-cell receptor repertoire by next-generation sequencing (BCR-Seq), as well as the analysis of serum antibodies against this antigen, using proteomics. The proteomic analysis is challenging since it necessitates the mapping of antigen-specific peptides to individual B-cell clones.ResultsThe PASA web server provides a robust computational platform for the analysis and integration of data obtained from proteomics of serum antibodies. PASA maps peptides derived from antibodies raised against a specific antigen to corresponding antibody sequences. It then analyzes and integrates proteomics and BCR-Seq data, thus providing a comprehensive characterization of the humoral response. The PASA web server is freely available at https://pasa.tau.ac.il and open to all users without a login requirement

Systematic Characterization and Comparative Analysis of the Rabbit Immunoglobulin Repertoire

<div><p>Rabbits have been used extensively as a model system for the elucidation of the mechanism of immunoglobulin diversification and for the production of antibodies. We employed Next Generation Sequencing to analyze Ig germline V and J gene usage, CDR3 length and amino acid composition, and gene conversion frequencies within the functional (transcribed) IgG repertoire of the New Zealand white rabbit (<i>Oryctolagus cuniculus</i>). Several previously unannotated rabbit heavy chain variable (VH) and light chain variable (VL) germline elements were deduced bioinformatically using multidimensional scaling and k-means clustering methods. We estimated the gene conversion frequency in the rabbit at 23% of IgG sequences with a mean gene conversion tract length of 59±36 bp. Sequencing and gene conversion analysis of the chicken, human, and mouse repertoires revealed that gene conversion occurs much more extensively in the chicken (frequency 70%, tract length 79±57 bp), was observed to a small, yet statistically significant extent in humans, but was virtually absent in mice.</p></div

Summary of 454 NGS data.

<p>Summary of 454 NGS data.</p

Diversification of the immunoglobulin repertoire – comparison of overall levels of nucleotide changes in the VH sequences across four species.

<p>The number of nucleotide differences from the nearest (assigned) VH germline derived from IgBLAST alignments as discussed in the text for rabbits.</p

Light chain germline gene usage.

<p>(A) Vκ and (B) Jκ germline usage of unique kappa light chain sequences in rabbits. Sample sizes of unique sequences: rab1 Bone marrow PC, N = 10,446; rab2 Bone marrow PC, N = 7,481; rab3 Bone marrow PC, N = 12,580. Germline gene IDs are as listed in the IMGT database.</p