# In-cell Automated Flash Oxidation (IC-AutoFox™) Protein Footprinting System

> **NIH NIH R44** · GENNEXT TECHNOLOGIES, INC. · 2023 · $1,165,416

## Abstract

The GenNext Phase II SBIR submssion entitled “In-cell Automated Flash
Oxidation (IC-AutoFox™) Protein Footprinting System,” is responsive to the
ackowledged need for new and improved tools for higher order structural analysis (HOS)
of biopharmaceuticals and membrane protein target studies. An emerging HOS analysis
technique is hydroxyl radical foot-printing (HRPF). HRPF involves the irreversible
labeling of a protein’s exterior by reaction with hydroxyl radicals with subsequent MS
analysis to identify the outer portions of the protein. The most widely used method for
generating OH radicals employs a quick burst of UV light, and is appropriately called fast
photochemical oxidation of proteins (FPOP).We have developed commercial solutions to
perform in vitro FPOP. The practice of applying the results of in vitro structural
experiments to authentic in vivo behavior has been brought into question.
Macromolecular crowding within a cell limits diffusion, thus altering reaction kinetics,
association rates of proteins, and protein-DNA interactions. These effects are not
observed while performing in vitro studies. Because of in vitro shortcomings, there has
been recent desire to extend the use of FPOP to whole cells in an in vivo manner.
 The practice of in vivo or in-cell FPOP (IC-FPOP) has been pioneered by Dr. Lisa
Jones of the University of Maryland. While showing great promise to address unmet
challenges in pharmaceutical research, reproducibility for IC-FPOP is challenged by
variability of intracellular background scavenging, cell-to-cell isolation irreproducibility,
and the mandatory use of hazardous, complicated, and poorly reproducible excimer
lasers. Collaborating with the Jones laboratory, our work will extend our innovative in
vitro laser-free FPOP technology to in vivo applications. GenNext Technologies is the
only company commercializing products for FPOP HOS analysis. Our goal is to convert
the IC-FPOP process from an academic research experiment into a valuable analytical
tool. Once simplified and transformed into a robust technique, we envision IC-FPOP to
enable cell-based assays to: paratope and epitope the interaction of mAb
biopharmaceuticals with their membrane targets; elucidate the dynamics of lead binding
to orthosteric or allosteric membrane targets; to reveal secondary messenger signaling
cascades of GPCR lead compounds; and to detect the impact of anti-neoplastics upon
targets such as kinases and growth factors.

## Key facts

- **NIH application ID:** 10589128
- **Project number:** 5R44GM137728-03
- **Recipient organization:** GENNEXT TECHNOLOGIES, INC.
- **Principal Investigator:** Scot Randy Weinberger
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $1,165,416
- **Award type:** 5
- **Project period:** 2020-04-01 → 2026-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10589128

## Citation

> US National Institutes of Health, RePORTER application 10589128, In-cell Automated Flash Oxidation (IC-AutoFox™) Protein Footprinting System (5R44GM137728-03). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10589128. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*