Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Autoantigen discovery with a synthetic human peptidome

Abstract

Immune responses targeting self-proteins (autoantigens) can lead to a variety of autoimmune diseases. Identification of these antigens is important for both diagnostic and therapeutic reasons. However, current approaches to characterize autoantigens have, in most cases, met only with limited success. Here we present a synthetic representation of the complete human proteome, the T7 peptidome phage display library (T7-Pep), and demonstrate its application to autoantigen discovery. T7-Pep is composed of >413,000 36-residue, overlapping peptides that cover all open reading frames in the human genome, and can be analyzed using high-throughput DNA sequencing. We developed a phage immunoprecipitation sequencing (PhIP-Seq) methodology to identify known and previously unreported autoantibodies contained in the spinal fluid of three individuals with paraneoplastic neurological syndromes. We also show how T7-Pep can be used more generally to identify peptide-protein interactions, suggesting the broader utility of our approach for proteomic research.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Construction and characterization of T7-Pep and the PhIP-Seq methodology.
Figure 2: Statistical analysis of PhIP-Seq data.
Figure 3: Validation of PhIP-Seq candidates.
Figure 4: PhIP-Seq can identify peptide-protein interactions.

References

  1. Graham, A.L. et al. Fitness correlates of heritable variation in antibody responsiveness in a wild mammal. Science 330, 662–665 (2010).

    CAS  Article  PubMed  Google Scholar 

  2. Faix, P.H. et al. Phage display of cDNA libraries: enrichment of cDNA expression using open reading frame selection. Biotechniques 36, 1018–1022 (2004).

    CAS  Article  PubMed  Google Scholar 

  3. Albert, M.L. & Darnell, R.B. Paraneoplastic neurological degenerations: keys to tumour immunity. Nat. Rev. Cancer 4, 36–44 (2004).

    CAS  Article  PubMed  Google Scholar 

  4. Wang, X. et al. Autoantibody signatures in prostate cancer. N. Engl. J. Med. 353, 1224–1235 (2005).

    CAS  Article  PubMed  Google Scholar 

  5. Anderson, K.S. et al. A protein microarray signature of autoantibody biomarkers for the early detection of breast cancer. J. Proteome Res. 10, 85–96 (2011).

    CAS  Article  PubMed  Google Scholar 

  6. Zacchi, P., Sblattero, D., Florian, F., Marzari, R. & Bradbury, A.R.M. Selecting open reading frames from DNA. Genome Res. 13, 980–990 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Kim, Y. et al. Identification of Hnrph3 as an autoantigen for acute anterior uveitis. Clin. Immunol. 138, 60–66 (2011).

    CAS  Article  PubMed  Google Scholar 

  8. Hughes, J.B., Hellmann, J.J., Ricketts, T.H. & Bohannan, B.J. Counting the uncountable: statistical approaches to estimating microbial diversity. Appl. Environ. Microbiol. 67, 4399–4406 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. Swann, J.B. & Smyth, M.J. Immune surveillance of tumors. J. Clin. Invest. 117, 1137–1146 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. Darnell, R.B. & Posner, J.B. Paraneoplastic syndromes involving the nervous system. N. Engl. J. Med. 349, 1543–1554 (2003).

    CAS  Article  PubMed  Google Scholar 

  11. Musunuru, K. & Kesari, S. Paraneoplastic opsoclonus-myoclonus ataxia associated with non-small-cell lung carcinoma. J. Neurooncol. 90, 213–216 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. Srivastava, S. & Chen, L. A two-parameter generalized Poisson model to improve the analysis of RNA-seq data. Nucleic Acids Res. 38, e170 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Consul, P. & Shoukri, M. Maximum likelihood estimation for the generalized poisson distribution. Comm. Statist. Theory Methods 13, 1533–1547 (1984).

    Article  Google Scholar 

  14. Bailey, T.L. & Elkan, C. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc. Int. Conf. Intell. Syst. Mol. Biol. 2, 28–36 (1994).

    CAS  PubMed  Google Scholar 

  15. Almeida, L.G. et al. CTdatabase: a knowledge-base of high-throughput and curated data on cancer-testis antigens. Nucleic Acids Res. 37, D816–D819 (2009).

    CAS  Article  PubMed  Google Scholar 

  16. Rimoldi, D. et al. Efficient simultaneous presentation of NY-ESO-1/LAGE-1 primary and nonprimary open reading frame-derived CTL epitopes in melanoma. J. Immunol. 165, 7253–7261 (2000).

    CAS  Article  PubMed  Google Scholar 

  17. Chen, Y.T. et al. Identification of multiple cancer/testis antigens by allogeneic antibody screening of a melanoma cell line library. Proc. Natl. Acad. Sci. USA 95, 6919–6923 (1998).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Blanco-Arias, P., Sargent, C.A. & Affara, N.A. The human-specific Yp11.2/Xq21.3 homology block encodes a potentially functional testis-specific TGIF-like retroposon. Mamm. Genome 13, 463–468 (2002).

    CAS  Article  PubMed  Google Scholar 

  19. Berglund, L. et al. A genecentric human protein atlas for expression profiles based on antibodies. Mol. Cell. Proteomics 7, 2019–2027 (2008).

    CAS  Article  PubMed  Google Scholar 

  20. Li, L., Hagopian, W.A., Brashear, H.R., Daniels, T. & Lernmark, A. Identification of autoantibody epitopes of glutamic acid decarboxylase in stiff-man syndrome patients. J. Immunol. 152, 930–934 (1994).

    CAS  PubMed  Google Scholar 

  21. Schwartz, H.L. et al. High-resolution autoreactive epitope mapping and structural modeling of the 65 kDa form of human glutamic acid decarboxylase. J. Mol. Biol. 287, 983–999 (1999).

    CAS  Article  PubMed  Google Scholar 

  22. Tanji, K. et al. TRIM9, a novel brain-specific E3 ubiquitin ligase, is repressed in the brain of Parkinson's disease and dementia with Lewy bodies. Neurobiol. Dis. 38, 210–218 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. Rual, J.-F. et al. Towards a proteome-scale map of the human protein-protein interaction network. Nature 437, 1173–1178 (2005).

    CAS  Article  PubMed  Google Scholar 

  24. Ciccia, A. et al. The SIOD disorder protein SMARCAL1 is an RPA-interacting protein involved in replication fork restart. Genes Dev. 23, 2415–2425 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Mer, G. et al. Structural basis for the recognition of DNA repair proteins UNG2, XPA, and RAD52 by replication factor RPA. Cell 103, 449–456 (2000).

    CAS  Article  PubMed  Google Scholar 

  26. Barlow, D.J., Edwards, M.S. & Thornton, J.M. Continuous and discontinuous protein antigenic determinants. Nature 322, 747–748 (1986).

    CAS  Article  PubMed  Google Scholar 

  27. Jin, L., Fendly, B.M. & Wells, J.A. High resolution functional analysis of antibody-antigen interactions. J. Mol. Biol. 226, 851–865 (1992).

    CAS  Article  PubMed  Google Scholar 

  28. Miyazaki, K. et al. Analysis of in vivo role of alpha-fodrin autoantigen in primary Sjogren's syndrome. Am. J. Pathol. 167, 1051–1059 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. Huang, M. et al. Detection of apoptosis-specific autoantibodies directed against granzyme B-induced cleavage fragments of the SS-B (La) autoantigen in sera from patients with primary Sjogren's syndrome. Clin. Exp. Immunol. 142, 148–154 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. Robbins, D.C., Cooper, S.M., Fineberg, S.E. & Mead, P.M. Antibodies to covalent aggregates of insulin in blood of insulin-using diabetic patients. Diabetes 36, 838–841 (1987).

    CAS  Article  PubMed  Google Scholar 

  31. Papachroni, K.K. et al. Autoantibodies to alpha-synuclein in inherited Parkinson's disease. J. Neurochem. 101, 749–756 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. Dalakas, M.C., Fujii, M., Li, M. & McElroy, B. The clinical spectrum of anti-GAD antibody-positive patients with stiff-person syndrome. Neurology 55, 1531–1535 (2000).

    CAS  Article  PubMed  Google Scholar 

  33. Derda, R. et al. Diversity of phage-displayed libraries of peptides during panning and amplification. Molecules 16, 1776–1803 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. Lamesch, P. et al. hORFeome v3.1: a resource of human open reading frames representing over 10,000 human genes. Genomics 89, 307–315 (2007).

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported in part by grants from the Department of Defense (W81XWH-10-1-0994 and W81XWH-04-1-0197) to S.J.E., and in part by the US National Institutes of Health (K08CA124804), The American Recovery and Reinvestment Act (3P30CA023100-25S8), Sontag Foundation Distinguished Scientist Award and a James S. McDonnell Foundation award to S.K. N.L.S. is a fellow of the Susan G. Komen for the Cure Foundation. S.J.E. is an investigator with the Howard Hughes Medical Institute. We would like to thank S. Gowrisankar, O. Iartchouk and L. Merrill for assistance with Illumina sequencing, and D. Šćepanović for statistical support.

Author information

Authors and Affiliations

Authors

Contributions

S.J.E. conceived the project, which was supervised by N.L.S. and S.J.E. Z.Z. designed the DNA sequences for synthesis. Oligo libraries were constructed by E.M.L. Cloning was performed by M.Z.L., M.A.M.G. and N.L.S. The T7-Pep, T7-NPep, and T7-CPep phage libraries were constructed by N.L.S. and characterized by N.L.S. and H.B.L. The PhIP-Seq protocol was developed and implemented by H.B.L. Clinical evaluations and patient sample acquisitions were performed by S.K. Statistical analysis of PhIP-Seq data was conceived by U.L. under the supervision of G.M.C. and implemented by H.B.L. PhIP-Seq candidates were confirmed by H.B.L. The RPA2 experiment was performed by A.C. The manuscript was prepared by H.B.L. and edited by N.L.S. and S.J.E.

Corresponding authors

Correspondence to Nicole L Solimini or Stephen J Elledge.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–3 and Supplementary Figs. 1–9 (PDF 5813 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Larman, H., Zhao, Z., Laserson, U. et al. Autoantigen discovery with a synthetic human peptidome. Nat Biotechnol 29, 535–541 (2011). https://doi.org/10.1038/nbt.1856

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.1856

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing