# Post-translational modification proteomics in 4D: Chemoenzymatic tools to map the dynamic spatial organization of eukaryotic signaling pathways

> **NIH NIH DP2** · UNIVERSITY OF WISCONSIN-MADISON · 2022 · $1,399,500

## Abstract

Project summary/abstract
 Spatial organization and temporal dynamics are inherent properties of eukaryotic cell signaling pathways.
Dynamic changes in the subcellular localization of proteins are programmed by post-translational modifications
(PTMs), such as phosphorylation and proteolysis, which occur in response to biological stimuli. Eukaryotic
signaling pathways rely on the introduction of PTMs to specific proteins to rapidly change protein function and
localization, enabling cells to respond to changing internal or environmental conditions. However, despite the
importance of spatial organization and temporal dynamics in biological signaling, current technologies are unable
to provide a systems-level experimental mapping of the dynamic subcellular localization of post-translationally
modified proteins. To meet this challenge, we propose to develop new methods for PTM proteomics with
subcellular spatial and temporal resolution by engineering genetically targetable, PTM-selective proximity
labeling enzymes. These enzymes will tag post-translationally modified proteins in specific subcellular locations,
enabling their enrichment and analysis with mass spectrometry-based proteomics experiments for mapping
PTMs with spatial and temporal resolution. We will initially focus on two pervasive PTMs, proteolysis and
phosphorylation, both of which play critical roles in numerous biological signaling pathways relevant to human
health and disease. We will apply protein engineering approaches to develop three distinct classes of enzymes
for spatiotemporally resolved capture of proteolytic neo-C termini, phosphoserine/phosphothreonine, and
phosphotyrosine, respectively. We will deploy these tools to dissect the spatiotemporal dynamics of proteolysis
during apoptosis; phosphorylation during growth factor signaling; and crosstalk between proteolysis and
phosphorylation during the cellular decision between life and death. The biological pathways and states that can
be probed with the tools that we will develop are nearly limitless, ensuring that they will have a broad and
transformative impact across the biomedical sciences. Completion of the proposed work will transform our
understanding of how cellular signaling unfolds across space and time and has the potential to reveal new
paradigms for therapeutic intervention in PTM-based signaling pathways.

## Key facts

- **NIH application ID:** 10471100
- **Project number:** 1DP2GM149548-01
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** Amy M Weeks
- **Activity code:** DP2 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $1,399,500
- **Award type:** 1
- **Project period:** 2022-09-01 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10471100

## Citation

> US National Institutes of Health, RePORTER application 10471100, Post-translational modification proteomics in 4D: Chemoenzymatic tools to map the dynamic spatial organization of eukaryotic signaling pathways (1DP2GM149548-01). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10471100. Licensed CC0.

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