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

Autor: Henson SN; The Translational Genomics Research Institute (TGen), Phoenix, AZ, USA., Elko EA; The Pathogen and Microbiome Institute, Northern Arizona University, Flagstaff, AZ, USA., Swiderski PM; DNA/RNA Synthesis Laboratory, Department of Molecular Medicine, Beckman Research Institute of the City of Hope, Duarte, CA, USA., Liang Y; DNA/RNA Synthesis Laboratory, Department of Molecular Medicine, Beckman Research Institute of the City of Hope, Duarte, CA, USA., Engelbrektson AL; The Translational Genomics Research Institute (TGen), Phoenix, AZ, USA., Piña A; The Pathogen and Microbiome Institute, Northern Arizona University, Flagstaff, AZ, USA., Boyle AS; The Translational Genomics Research Institute (TGen), Phoenix, AZ, USA., Fink Z; The Pathogen and Microbiome Institute, Northern Arizona University, Flagstaff, AZ, USA., Facista SJ; The Translational Genomics Research Institute (TGen), Phoenix, AZ, USA., Martinez V; The Pathogen and Microbiome Institute, Northern Arizona University, Flagstaff, AZ, USA., Rahee F; The Translational Genomics Research Institute (TGen), Phoenix, AZ, USA., Brown A; The Pathogen and Microbiome Institute, Northern Arizona University, Flagstaff, AZ, USA., Kelley EJ; The Translational Genomics Research Institute (TGen), Flagstaff, AZ, USA., Nelson GA; The Translational Genomics Research Institute (TGen), Flagstaff, AZ, USA., Raspet I; The Pathogen and Microbiome Institute, Northern Arizona University, Flagstaff, AZ, USA., Mead HL; The Translational Genomics Research Institute (TGen), Flagstaff, AZ, USA., Altin JA; The Translational Genomics Research Institute (TGen), Flagstaff, AZ, USA. jaltin@tgen.org., Ladner JT; The Pathogen and Microbiome Institute, Northern Arizona University, Flagstaff, AZ, USA. jason.ladner@nau.edu.
Jazyk: angličtina
Zdroj: Nature protocols [Nat Protoc] 2023 Feb; Vol. 18 (2), pp. 396-423. Date of Electronic Publication: 2022 Nov 16.
DOI: 10.1038/s41596-022-00766-8
Abstrakt: 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.
(© 2022. Springer Nature Limited.)
Databáze: MEDLINE
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