# Multiplex labeling chemistry methods for protein footprinting

> **NIH NIH R01** · CASE WESTERN RESERVE UNIVERSITY · 2024 · $378,804

## Abstract

Abstract
Protein footprinting (PF) is a powerful medium resolution structural biology technique for
assessing protein structure and dynamics that relies on “bottom-up” mass spectrometry (MS) to
detect, identify, and quantitatively analyze the small (5-15 residue) peptides that are generated in
protease-based workflows. Early on hydrogen deuterium exchange (HDX) led the way, later
advances in irreversible reagent development, such as hydroxyl radical footprinting (HRF)
mediated by radiolysis; or methods utilizing photolysis of peroxide, and recently plasma, have
been introduced and refined. As a result, PF is routinely applied in understanding the effects of
protein-ligand binding on higher order interactions in solution, even for large macromolecules like
antibodies, large molecular complexes, and membrane proteins; all of which are important drug
targets and biological machines. However, state of the art HRF-based PF studies typically report
data from only <20% of the possible sites within the above peptides, limiting the overall impact.
In part this is due to the high reactivity of sulfur containing (Met in particular) and aromatic residues
relative to many others, creating dynamic range issues for simultaneously detecting high and low
abundant species in the same experiment. In this proposal, responsive to PAR-19-253, we
propose a range of novel labeling, biophysics, and mass spectrometry methods, based on
radical activated trifluoromethylation based chemistries, to provide multiplex, high-
resolution labeling and mass spectrometry analysis workflows to maximize value and
impact of PF studies, with readouts from 50-100% of accessible side chains within the
relevant peptides. The project leverages our advanced synchrotron radiolysis platform and our
validation and collaboration strategies will extend the results to major PF platforms such as:
radiolysis, photolysis, and plasma based HRF.
In Aim 1 (months 0-20), we will benchmark the chemistry of hydroxyl radical induced TFM,
understanding its side chain-based reactivity compared to OH radical by examining amino acid
and peptide based reactivity for a variety of available TFM reagents. Initial developments of these
TFM labeling approaches leverage our advanced synchrotron radiolysis platform for HRF, but we
will validate the method using both radiolysis and photolysis, and finding optimum conditions for
expanding PF coverage while optimizing dynamic range of labeling. Milestones: Modify ~16/20
residues in a one-pot reaction (vs ~12/20 today). Dynamic range of ~100 or less in separate +16
(-O) and +68 (-CF3) channels (vs. 1000-today in one +16 (-O) channel). In Aim 2 (months 20-
40), we will extend our findings that the Langlois reagent is a promising candidate for hydroxyl
radical induced TFM and probing protein structure in a quantitative way using structurally
understood and PF tractable calmodulin and estrogen receptor as benchmarked targets.
Milestone: Optimize and validate a simple, easy to...

## Key facts

- **NIH application ID:** 10812459
- **Project number:** 5R01GM141078-04
- **Recipient organization:** CASE WESTERN RESERVE UNIVERSITY
- **Principal Investigator:** Janna Kiselar
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $378,804
- **Award type:** 5
- **Project period:** 2021-05-01 → 2025-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10812459

## Citation

> US National Institutes of Health, RePORTER application 10812459, Multiplex labeling chemistry methods for protein footprinting (5R01GM141078-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10812459. Licensed CC0.

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