# In-Cell Radical Dosimetry (ICRD) for improved in vivo Fast Photo-oxidation of Proteins Hydroxyl Radical Protein Footprinting

> **NIH NIH R43** · GENNEXT TECHNOLOGIES, INC. · 2020 · $261,509

## Abstract

The GenNext Phase I SBIR submssion entitled “In-Cell Radical Dosimetry
(ICRD) for improved in vivo FPOP HRPF” 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 [13].
 The practice of in vivo or in-cell FPOP (IC-FPOP) has been pioneered by Dr. Lisa
Jones of the University of Maryland [13, 26-28]. While showing great promise to
address unmet challenges in pharmaceutical research, reproducibility for IC-FPOP is
challenged by variability of intracellular background scavenging and cell-to-cell isolation
irreproducibility. Collaborating with the Jones laboratory, our work will extend our
innovative in vitro radical dosimetry technology to in vivo radical scavenging. 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 orthosteric /
allosteric anti-neoplastics upon targets such as kinases and growth factors.

## Key facts

- **NIH application ID:** 10009765
- **Project number:** 1R43GM137728-01
- **Recipient organization:** GENNEXT TECHNOLOGIES, INC.
- **Principal Investigator:** Scot Randy Weinberger
- **Activity code:** R43 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $261,509
- **Award type:** 1
- **Project period:** 2020-04-01 → 2021-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10009765

## Citation

> US National Institutes of Health, RePORTER application 10009765, In-Cell Radical Dosimetry (ICRD) for improved in vivo Fast Photo-oxidation of Proteins Hydroxyl Radical Protein Footprinting (1R43GM137728-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10009765. Licensed CC0.

---

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