The binding specificities of an individual’s antibody repertoire contain a wealth of biological information. They harbor evidence of environmental exposures, allergies, ongoing or emerging autoimmune disease processes, and responses to immunomodulatory therapies, for example. Highly multiplexed methods to comprehensively interrogate antibody-binding specificities have therefore emerged in recent years as important molecular tools. Here, we provide a detailed protocol for performing ‘phage immunoprecipitation sequencing’ (PhIP-Seq), which is a powerful method for analyzing antibody-repertoire binding specificities with high throughput and at low cost. The methodology uses oligonucleotide library synthesis (OLS) to encode proteomic-scale peptide libraries for display on bacteriophage. These libraries are then immunoprecipitated, using an individual’s antibodies, for subsequent analysis by high-throughput DNA sequencing. We have used PhIP-Seq to identify novel self-antigens associated with autoimmune disease, to characterize the self-reactivity of broadly neutralizing HIV antibodies, and in a large international cross-sectional study of exposure to hundreds of human viruses. Compared with alternative array-based techniques, PhIP-Seq is far more scalable in terms of sample throughput and cost per analysis. Cloning and expression of recombinant proteins are not required (versus protein microarrays), and peptide lengths are limited only by DNA synthesis chemistry (up to 90-aa (amino acid) peptides versus the typical 8- to 12-aa length limit of synthetic peptide arrays). Compared with protein microarrays, however, PhIP-Seq libraries lack discontinuous epitopes and post-translational modifications. To increase the accessibility of PhIP-Seq, we provide detailed instructions for the design of phage-displayed peptidome libraries, their immunoprecipitation using serum antibodies, deep sequencing–based measurement of peptide abundances, and statistical determination of peptide enrichments that reflect antibody–peptide interactions. Once a library has been constructed, PhIP-Seq data can be obtained for analysis within a week.
Your institute does not have access to this article
Open Access articles citing this article.
Scientific Reports Open Access 28 January 2021
Nature Communications Open Access 22 January 2021
Mobile DNA Open Access 31 December 2019
Subscribe to Nature+
Get immediate online access to the entire Nature family of 50+ journals
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Larman, H. B. et al. Autoantigen discovery with a synthetic human peptidome. Nat. Biotechnol. 29, 535–541 (2011).
Larman, H. B. et al. PhIP-Seq characterization of autoantibodies from patients with multiple sclerosis, type 1 diabetes and rheumatoid arthritis. J. Autoimmun. 43, 1–9 (2013).
Larman, H. B. et al. Cytosolic 5′-nucleotidase 1A autoimmunity in sporadic inclusion body myositis. Ann. Neurol. 73, 408–418 (2013).
Finton, K. A. et al. Ontogeny of recognition specificity and functionality for the broadly neutralizing anti-HIV antibody 4E10. PLoS Pathog. 10, e1004403 (2014).
Xu, G. J. et al. Viral immunology. Comprehensive serological profiling of human populations using a synthetic human virome. Science 348, aaa0698 (2015).
Kosuri, S. & Church, G. M. Large-scale de novo DNA synthesis: technologies and applications. Nat. Methods 11, 499–507 (2014).
Atak, A. et al. Protein microarray applications: autoantibody detection and posttranslational modification. Proteomics 16, 2557–2569 (2016).
Yu, X. et al. Multiplexed nucleic acid programmable protein arrays. Theranostics 7, 4057–4070 (2017).
Henkel, S., Wellhausen, R., Woitalla, D., Marcus, K. & May, C. Epitope mapping using peptide microarray in autoantibody profiling. Methods Mol. Biol. 1368, 209–224 (2016).
Finton, K. A. et al. Autoreactivity and exceptional CDR plasticity (but not unusual polyspecificity) hinder elicitation of the anti-HIV antibody 4E10. PLoS Pathog. 9, e1003639 (2013).
Xu, G.J. et al. Systematic autoantigen analysis identifies a distinct subtype of scleroderma with coincident cancer. Proc. Natl. Acad. Sci. USA 113, E7526-E7534 (2016).
Zhu, H., Luo, H., Yan, M., Zuo, X. & Li, Q. Z. Autoantigen microarray for high-throughput autoantibody profiling in systemic lupus erythematosus. Genomics Proteomics Bioinformatics 13, 210–218 (2015).
Miersch, S. & LaBaer, J. Nucleic acid programmable protein arrays: versatile tools for array-based functional protein studies. Curr. Protoc. Protein Sci. Chapter 27, Unit 27.2 (2011).
Zhu, J. et al. Protein interaction discovery using parallel analysis of translated ORFs (PLATO). Nat. Biotechnol. 31, 331–334 (2013).
Larman, H. B., Liang, A. C., Elledge, S. J. & Zhu, J. Discovery of protein interactions using parallel analysis of translated ORFs (PLATO). Nat. Protoc. 9, 90–103 (2014).
Jhaveri, D. T. et al. Using quantitative seroproteomics to identify antibody biomarkers in pancreatic cancer. Cancer Immunol. Res. 4, 225–233 (2016).
MacConaill, L. E. et al. Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing. BMC Genomics 19, 30 (2018).
The development of the PhIP-Seq technology platform has been an ongoing collaboration with S.J. Elledge of the Harvard Medical School Genetics Department and the Howard Hughes Medical Institute. Special thanks to T.M. Shi (Department of Art as Applied to Medicine, Johns Hopkins School of Medicine) for creating the artwork used in Fig. 1. Recent improvements in the PhIP-Seq methodology were supported under a U24 Resource-Related Research Projects Cooperative Agreement awarded by the NIH (U24AI118633 to H.B.L. and S.J. Elledge), a grant from the Jerome L. Greene Foundation (to H.B.L. and A.N.B.), and a grant from the Sjögren’s Syndrome Foundation (to H.B.L. and A.N.B.). A.N.B. received support from NIH grant R01DE012354 (Rosen, PI).
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Key references using this protocol
1. Larman, H. B. et al. Nat. Biotech. 29, 535–541 (2011) https://doi.org/10.1038/nbt.1856
2. Larman, H. B. et al. Ann. Neurol. 73, 408–418 (2013) https://doi.org/10.1002/ana.23840
3. Xu, G. J. et al. Science 384, aaa0698 (2015) https://doi.org/10.1126/science.aaa0698
About this article
Cite this article
Mohan, D., Wansley, D.L., Sie, B.M. et al. PhIP-Seq characterization of serum antibodies using oligonucleotide-encoded peptidomes. Nat Protoc 13, 1958–1978 (2018). https://doi.org/10.1038/s41596-018-0025-6
Population-wide diversity and stability of serum antibody epitope repertoires against human microbiota
Nature Medicine (2021)
Nature Communications (2021)
Scientific Reports (2021)
Nature Medicine (2021)
Mobile DNA (2020)