Next-generation sequencing and protein mass spectrometry for the comprehensive analysis of human cellular and serum antibody repertoires Jason J Lavinder 1,2 , Andrew P Horton 3,4 , George Georgiou 1,2,3,4,5 and Gregory C Ippolito 5 Recent developments of high-throughput technologies are enabling the molecular-level analysis and bioinformatic mining of antibody-mediated (humoral) immunity in humans at an unprecedented level. These approaches explore either the sequence space of B-cell receptor repertoires using next- generation deep sequencing (BCR-seq), or the amino acid identities of antibody in blood using protein mass spectrometry (Ig-seq), or both. Generalizable principles about the molecular composition of the protective humoral immune response are being defined, and as such, the field could supersede traditional methods for the development of diagnostics, vaccines, and antibody therapeutics. Three key challenges remain and have driven recent advances: (1) incorporation of innovative techniques for paired BCR-seq to ascertain the complete antibody variable-domain VH:VL clonotype, (2) integration of proteomic Ig-seq with BCR-seq to reveal how the serum antibody repertoire compares with the antibody repertoire encoded by circulating B cells, and (3) a demand to link antibody sequence data to functional meaning (binding and protection). Addresses 1 Department of Chemical Engineering, University of Texas at Austin, Austin, TX 78712-1062, USA 2 Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712-1062, USA 3 Center for Systems & Synthetic Biology, University of Texas at Austin, Austin, TX 78712-1062, USA 4 Department of Biomedical Engineering, University of Texas at Austin, Austin, TX 78712-1062, USA 5 Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712-1062, USA Corresponding author: Ippolito, Gregory C (gci@mail.utexas.edu) Current Opinion in Chemical Biology 2015, 24:112–120 This review comes from a themed issue on Omics Edited by Benjamin F Cravatt and Thomas Kodadek http://dx.doi.org/10.1016/j.cbpa.2014.11.007 1367-5931/Published by Elsevier Ltd. Introduction Since the landmark discovery of antibody (or immuno- globulin, Ig) in blood serum more than 100 years ago, we now know conclusively that serum is composed of a complex spectrum of distinct antibodies (is polyclonal) and is generated by individual B-cell clones through extraordinary modes of genetic recombination, diversifi- cation, and selection by antigen (antibody generator) according to rules outlined in the paradigmatic ‘clonal selection theory’. Remarkably, however, there had been no way to identify, and determine the relative concen- trations, of the monoclonal antibodies (mAbs) that com- prise the serum polyclonal pool elicited in response to vaccination or natural infection, until recently [1  ,4  ,5]. Understanding the composition of the antigen-specific serum antibody protein repertoire, the properties (e.g., affinities, epitopes recognized) of the respective Ig, and finally, the relationship between circulating Ig and the presence of clonally expanded peripheral B cells is pro- foundly important for the comprehensive understanding of humoral antibody responses. The current era of modern genomics and proteomics is providing extraordinary new tools for examining antibody repertoires. Next Generation Sequencing (NGS) allows millions of B cell receptor (BCR) sequences to be obtained in a single experiment, and NGS approaches to studying the human antibody repertoire [6] not only aim to aid in the discovery of elite antibodies potentially useful as therapeutics, but also to comprehensively cat- alogue the antibody sequences that are elicited during an adaptive immune response [7]. Previously a limitation with NGS, the ability to obtain the endogenous variable heavy and light chain (VH:VL) pairs within NGS datasets is now feasible [1  ,8  ,9,10]. This paired VH:VL se- quencing represents a major breakthrough in BCR reper- toire analysis, obviating the need for multiplexed screening to identify functionally paired VH and VL. NGS has also provided a stepping stone to the direct characterization of serum antibodies (Ig) using NGS database-driven high-resolution protein mass spectrom- etry [1  ,2,3,4  ], providing a direct means to comprehen- sive delineation of the antibody repertoire. Two antibody repertoires: the cellular and the serological B cells, serum immunoglobulin, and persistence of the antibody repertoire The mechanisms of antibody diversification and B-cell differentiation have been expertly reviewed elsewhere Available online at www.sciencedirect.com ScienceDirect Current Opinion in Chemical Biology 2015, 24:112–120 www.sciencedirect.com