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Autoantigen discovery with a synthetic human peptidome - PubMed

  • ️Sat Jan 01 2011

Autoantigen discovery with a synthetic human peptidome

H Benjamin Larman et al. Nat Biotechnol. 2011.

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.

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Figures

Figure 1
Figure 1. Construction and characterization of T7-Pep and the PhIP-Seq methodology

(a) The T7-Pep library is made from 413,611 DNA sequences encoding 36 amino acid peptide tiles that span 24,239 unique ORFs from build 35.1 of the human genome. Each tile overlaps its neighbors by seven amino acids on each side. (b) The DNA sequences from (a) were printed as 140-mer oligos on releasable DNA microarrays. (i) After oligo release, the DNA was PCR-amplified and cloned into a FLAG-expressing derivative of the T7Select 10-3b mid copy phage display system. (ii) The T7-Pep library is mixed with patient samples containing autoantibodies. (iii) Antibodies and bound phage are captured on magnetic protein A/G coated beads. (iv) DNA from the immunoprecipitated phage is recovered and (v) library inserts are PCR-amplified with sequencing adapters. A single nucleotide change (arrow) is introduced for multiplex analysis. (c) Pie chart showing results of plaque sequencing of 71 phage from T7-Pep Pool 1 and T7-CPep Pool 1. (d) Histogram plot showing results from Illumina sequencing of T7-Pep. 78% of the total area lies between the vertical red lines at 10 and 100 reads, demonstrating the relative uniformity of the library. Representation of each subpool in T7-Pep (inset) compared to expected (horizontal red line).

Figure 2
Figure 2. Statistical analysis of PhIP-Seq data

(a) Scatter plot comparing sequencing reads from T7-Pep input library and from Patient A immunoprecipitated (IP) phage (Pearson coefficient = 0.435; P ~= 0). Highlighted are all clones with an input abundance of 50 reads (red), and all clones with an input abundance of 100 reads (blue). The target of the SAPK4 control antibody is highlighted in green. (b) Histogram plot of sequencing reads from the data highlighted in (a) with corresponding colors. The curves are fit with a generalized Poisson (GP) distribution. Pmf is the probability mass function of the corresponding GP distribution and x is the number of IP sequencing reads. (c) Plots of lambda and theta for each input abundance, calculated using the method of Consul et al. Lambda is regressed to its average value (black dashed line) and theta is linearly regressed (red dashed curve). (d) Scatter plot comparing clone enrichment significances (as –Log10 p-value) from two independent PhIP-Seq experiments using CSF from Patient A. Red dashed line shows the cutoff for considering a clone to be significantly enriched, and the SAPK4 control antibody target is highlighted in green.

Figure 3
Figure 3. Validation of full-length PhIP-Seq candidates

(a) Western blot with CSF from Patient A, staining for full-length TGIF2LX-GFP expressed in 293T cells by transient transfection. Bands corresponding to TGIF2LX-GFP are denoted by an arrow. (b) ClustalW alignment of the seven significantly enriched hypothetical protein LOC26080 peptides, and the nine-element MEME-generated recognition motif. (c) Western blot with CSF from Patient B, staining for indicated full-length proteins expressed in 293T cells by transient transfection. (d) Bar graph of –Log10 p-values of enrichment for the indicated TGIF2LX peptides by the three patients. (e) Immunoprecipitation of the GAD65-GFP from (c) by CSF from Patient B (but not Patient A). (f) Western blot with CSF from Patient C, staining for indicated full-length proteinss expressed in 293T cells by transient transfection. (g) Phage lysates from candidate T7 clones were spotted directly onto nitrocellulose membranes, which were subsequently immunoblotted with patient CSF.

Figure 4
Figure 4. PhIP-Seq can identify protein-protein interactions

GST-RPA2 was used to precipitate phage from the T7-Pep library on magnetic glutathione beads. (a) –Log10 p-values of enrichment were calculated using the generalized Poisson method. Clones are arranged in increasing input abundance from left to right. The experiment identified two of the known RPA2 binding partners SMARCAL1 (P < 10-14) and UNG2 (P < 10-5), highlighted in red. (b) Dependence of peptide enrichment on integrity of RPA2 binding motif. Phage peptides containing the RPA2-binding motif (underlined) are shown next to their –Log10 p-value of enrichment.

Comment in

  • Human peptidome display.

    Robinson WH, Steinman L. Robinson WH, et al. Nat Biotechnol. 2011 Jun 7;29(6):500-2. doi: 10.1038/nbt.1888. Nat Biotechnol. 2011. PMID: 21654669 Free PMC article.

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