PhIP-Seq characterization of serum antibodies using oligonucleotide-encoded peptidomes

Abstract

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.

LINE-1 ORF2p Expression is Nearly Imperceptible in Human Cancers

“…To assess the specificity of antibodies for L1Hs, we employed phage-display immunoprecipitation sequencing (PhIP-seq) [45][46][47]. We obtained all annotated repeats in the human genome from RepeatMasker and constructed a representational phage-display library which was used in combination with a previously constructed pan human proteome library [48].…”

“…The PhIP-Seq assay was described previously [47]. Approximately 100 ng of each mAb was added to the combined T7 bacteriophage human peptidome library (unique genome and repetitive element sublibrary addition, 1 x 105 plaque forming units for each phage clone in each library) and incubated with rotation overnight at 4 • C in deep 96-well plates in 1 mL total volume of phosphate-buffered saline.…”

Protease Activity Profiling Via Programmable Phage Display

“…Subcloning the Human Peptidome Library into the T7-SEPARATE Vector. T7-Pep2, 32 a complete human proteome library was restriction cloned into the T7-SEPARATE vector using the EcoRI/XhoI sites. 32,33 The library was packaged in vitro using the T7Select Packaging Kit (Millipore Sigma) and expanded by plate lysate amplification to obtain an average clonal representation of ~100 plaques per peptide.…”

“…T7-Pep2, 32 a complete human proteome library was restriction cloned into the T7-SEPARATE vector using the EcoRI/XhoI sites. 32,33 The library was packaged in vitro using the T7Select Packaging Kit (Millipore Sigma) and expanded by plate lysate amplification to obtain an average clonal representation of ~100 plaques per peptide. To select for AviTagdisplaying peptides, the initial expansion of this library was immobilized on streptavidin beads, washed to remove unbound phage particles (to remove prematurely truncated or out-of-frame peptides which will lack the downstream AviTag), digested with enterokinase, and re-expanded by plate lysate amplification at >100-times representation per peptide.…”

Exploration of Anti-Yo and Anti-Hu paraneoplastic neurological disorders by PhIP-Seq reveals a highly restricted pattern of antibody epitopes

“…Several statistical approaches for analyzing PhIP-Seq data have been published, primarily based on Generalized Poisson (GP) or Negative Binomial (NB) count models fit to the distribution of unselected or input library phage populations. 22,23 More recently, an algorithm leveraging z-scores of phage counts relative to AG bead only "mock" IPs was developed to account for non-specific phage binding to IP reagents and substrates. 24 These approaches were developed to analyze datasets generated from single rounds of immunoprecipitation and were optimized for sensitivity to low abundance phage.…”