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

Area of Science:

• Biochemistry
• Molecular Biology
• Immunology

Background:

• High-throughput proteomic assays are crucial for understanding molecular interactions.
• Current methods for epitope mapping can be time-consuming and costly.
• There is a need for efficient platforms to analyze antibody reactivity.

Purpose of the Study:

• To introduce PepSeq, an in vitro platform for building and conducting highly multiplexed proteomic assays.
• To describe the protocol for synthesizing DNA-barcoded peptide libraries and their application in serology.
• To demonstrate PepSeq's utility for fast and cost-effective epitope-level antibody reactivity analysis.

Main Methods:

• Design and synthesis of DNA-barcoded peptide libraries from DNA oligonucleotides.
• Covalent linking of synthesized peptides to corresponding cDNA tags.
• Performing highly multiplexed serological assays using these peptide/DNA conjugates.
• Leveraging high-throughput sequencing for data acquisition and analysis.

Main Results:

• PepSeq enables epitope-level analysis of antibody reactivity across hundreds of thousands of peptides.
• The platform requires minimal sample input (<1 µl of serum or plasma).
• Library synthesis takes 1-2 weeks, with serological assays and sequencing data generated in approximately 4 days.

Conclusions:

• PepSeq provides a fast, cost-effective, and highly multiplexed approach for proteomic assays.
• The platform facilitates fine-scale mapping of antibody epitopes and pathogen exposure history determination.
• Available software tools, including PepSIRF, support library design and data analysis.