PepSeq Provides Massive Platform for Antibody Analyses

Technology developed at TGen described in a new study.


PHOENIX, Ariz. — December 5, 2022 — With less than one microliter of blood plasma or serum, researchers can now use a customizable lab platform called PepSeq to observe how the tiny sample interacts with hundreds of thousands of protein building blocks.

The instructions for using PepSeq to analyze antibody responses, developed by scientists at the Translational Genomics Research Institute (TGen), part of City of Hope, and Northern Arizona University (NAU), was described in Nature Protocols.

The technology has already been used to analyze antibodies generated by COVID-19 vaccines, among other applications, said John Altin, Ph.D., Assistant Professor in TGen’s Pathogen Genomics and Integrated Cancer Genomics Divisions and one of PepSeq’s creators.

The platform starts with designing a “library” of peptides of interest—short strings of amino acids that are the building blocks of proteins. Each peptide is then linked to a unique DNA tag, which allows the scientists to pinpoint which peptides are being targeted by which antibodies (or other proteins) in a sample.

Traditional analyses of this type can usually only track the response of an antibody to one peptide target at a time, but PepSeq expands this capability to thousands or hundreds of thousands of targets.

“The technology for linking peptides to DNA traces back a long way, but it was really the development of next-generation sequencing that allowed us to repurpose that peptide-DNA linkage and use it to generate assays on custom content at scale, like the SARS-CoV-2 virus,” said Altin.

It takes about two months to create a PepSeq library, with most of that time involving the design stages and only two to three weeks of lab work, Altin explained.

For COVID-19, “it was probably two or three months from the initial sign that this was going to be a serious global issue to us having some data on that very same, previously unknown, new virus,” Altin recalled. “That kind of rapid turnaround and the ability to study a completely new target like SARS-CoV-2 is one of the attractive features of the technology.”

The researchers want to expand the PepSeq system to create even longer peptides for the libraries. Right now, most of the peptides are about 30 amino acids long, although the scientists have created ones that are 64 amino acids long.

“But really I think the next frontier would be taking that up to the hundreds of amino acids and starting to make full-length proteins or domains, which may allow us to identify aspects of the response that we are currently unable to see,” Altin said.

For instance, some antibodies recognize a viral protein by the protein’s folding structure, which would require a full protein that includes that folding shape, he explained.

Altin and his colleagues, including Jason Ladner, Ph.D., at NAU (co-senior author on the protocols paper), have been using the platform to explore the full range of viruses that infect humans and looking at ways that PepSeq could be developed as a surveillance tool to detect when animal viruses cross over into people.

The researchers also have a project underway to study antibody responses to the repertoire of bacteria that infect people and one centered on personalized cancer immunotherapies.

“This technology is very well adapted to screening across that kind of broad diversity,” Altin said.

Research funding for the study comes from the NIAID (U24AI152172, U24AI152172-01S1 and U24AI152172-IOF); the National Institute on Minority Health and Health Disparities of the National Institutes of Health (U54MD012388); and the State of Arizona Technology and Research Initiative Fund (TRIF, administered by the Arizona Board of Regents, through Northern Arizona University).

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