PepSeq: a fully in vitro platform for highly multiplexed serology using customizable DNA-barcoded peptide libraries

Abstract

PepSeq is an in vitro platform for building and conducting highly multiplexed proteomic assays against customizable targets by using DNA-barcoded peptides. Starting with a pool of DNA oligonucleotides encoding peptides of interest, this protocol outlines a fully in vitro and massively parallel procedure for synthesizing the encoded peptides and covalently linking each to a corresponding cDNA tag. The resulting libraries of peptide/DNA conjugates can be used for highly multiplexed assays that leverage high-throughput sequencing to profile the binding or enzymatic specificities of proteins of interest. Here, we describe the implementation of PepSeq for fast and cost-effective epitope-level analysis of antibody reactivity across hundreds of thousands of peptides from <1 µl of serum or plasma input. This protocol includes the design of the DNA oligonucleotide library, synthesis of DNA-barcoded peptide constructs, binding of constructs to sample, preparation for sequencing and data analysis. Implemented in this way, PepSeq can be used for a number of applications, including fine-scale mapping of antibody epitopes and determining a subject’s pathogen exposure history. The protocol is divided into two main sections: (i) design and synthesis of DNA-barcoded peptide libraries and (ii) use of libraries for highly multiplexed serology. Once oligonucleotide templates are in hand, library synthesis takes 1–2 weeks and can provide enough material for hundreds to thousands of assays. Serological assays can be conducted in 96-well plates and generate sequencing data within a further ~4 d. A suite of software tools, including the PepSIRF package, are made available to facilitate the design of PepSeq libraries and analysis of assay data.

Introduction

Antibodies are important clinical, immunological and epidemiological biomarkers^1,2. They are stable circulating molecules whose production requires multiple layers of cellular cooperation and genetic diversification with antigen-driven selection, making them highly specific reporters of both the inciting agent and the host’s immune capacity. At the same time, the enormous diversity of the antibody repertoire—and its possible antigenic targets—poses a challenge: traditional assays are able to quantify only a single antibody specificity per unit volume of material and thus are unable to provide a comprehensive view of the response from a typical sample. Instead, the next generation of immunomonitoring requires highly multiplexed but cost-effective approaches capable of resolving antibody reactivity across large numbers of targets simultaneously by using a small sample volume.

Traditional serological assays such as ELISA use immobilized antigen (e.g., peptides, proteins and whole pathogens) and enzymatic or fluorescent detection to quantify antibody reactivity against a single target at a time³. Although they can be highly quantitative, these assays are limited in their ability to provide breadth and resolution, because each additional target of interest requires the consumption of additional serum or plasma, which is often limiting, as well as other associated reagents, which can quickly inflate cost. To obtain high sensitivity, these assays also commonly use large, complex antigens with the potential for cross-reactivity among closely related targets. The use of encoded beads has expanded the multiplexity of serological assays to the range of 5–500 simultaneous antigens^4,5, but this remains incommensurate with the diversity of natural antibody responses. In addition, high-density peptide array technologies have been developed that allow hundreds to hundreds of thousands of peptides to be assayed simultaneously^6,7; however, these approaches have not yet achieved widespread uptake, probably in part because the need to manufacture and/or process one array for each sample can be cost prohibitive for larger-scale experiments.

In contrast, highly multiplexed serology can be achieved cost-effectively by solution-phase assays in which antibodies are probed with highly diverse libraries of DNA-associated antigens, and binding is then quantified by measuring the relative abundance of each peptide by using high-throughput sequencing. Populations of phage particles displaying defined libraries of peptides on their surface represent a successful embodiment of this approach^8,9 and have been used to profile reactivity across the human proteome¹⁰ as well as the proteomes of all human-infecting viruses^8,11. Here, we describe a method (PepSeq) predicated on the same general principle as phage display but involving a much simpler molecular construct—a covalent adduct of peptide and cDNA. A derivative of mRNA display, this process uses bulk enzymatic transcription and translation followed by puromycin-mediated intramolecular coupling between the peptide and its encoding nucleic acid sequence¹² to build high-complexity libraries representing antigen sets of interest that can be used to deeply profile polyclonal antibody repertoires. Advantages of this approach include the reduced potential for non-specific background binding, as well as the ability to construct libraries for highly multiplexed serology assays entirely in vitro (Fig. 1)…