# Multi-Wavelength Fluorescence Radical Dosimetry for Real-Time Assessment of Protein Footprinting Radical Yield

> **NIH NIH R43** · GENNEXT TECHNOLOGIES, INC. · 2021 · $251,384

## Abstract

The GenNext Phase I SBIR submssion entitled “Multi-Wavelength Fluorescence
Radical Dosimetry for Real-Time Assessment of Protein Footprinting Radical Yield” 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. We have
developed commercial solutions to perform HRPF. Recently a new and valuable
footprinting technique that relies upon trifluoromethyl (TFM) radicals created by OH
radical attack of aqueous sodium triflinate has been developed that shows great promise
when combined with HRPF. TFM protein footprinting (TFMPF) is highly complementary
to HRPF, as TFMPF effectively labels amino acid residues that are relatively “silent” to
OH radical attack. When used together, HRPF and TFMPF provide substantial coverage
and detection of solvent accessible residues, and as such represent a transformative
improvement to biopharmaceutical HOS assessment.
 The practice of TFMPF has been pioneered by Professor Michael Gross of
Washington University, St. Louis, Mo. While showing great promise to address unmet
challenges in pharmaceutical research, reproducibility for TFMPF is challenged by
variability of background scavenging. Collaborating with the Gross laboratory, our work
will extend our innovative HRPF radical dosimetry technology to TFMPF. GenNext
Technologies is the only company commercializing products for HRPF HOS analysis.
Our goal is to convert the combined use of HRPF and TFMPF process from an
academic research experiment into a valuable analytical tool. Once simplified and
transformed into a robust technique, we envision the facile combination of HRPF and
TFMPF to enable: 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:** 10250755
- **Project number:** 1R43GM142351-01
- **Recipient organization:** GENNEXT TECHNOLOGIES, INC.
- **Principal Investigator:** Scot Randy Weinberger
- **Activity code:** R43 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $251,384
- **Award type:** 1
- **Project period:** 2021-05-01 → 2023-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10250755

## Citation

> US National Institutes of Health, RePORTER application 10250755, Multi-Wavelength Fluorescence Radical Dosimetry for Real-Time Assessment of Protein Footprinting Radical Yield (1R43GM142351-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10250755. Licensed CC0.

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