# Chemical Approaches to Characterizing Protein Post-translational Modifications by Mass Spectrometry

> **NIH NIH R35** · WAYNE STATE UNIVERSITY · 2021 · $56,384

## Abstract

Summary
Most biological events in the cell are mediated at some level by protein post-translational modifications. For
example, aberrant protein phosphorylation catalyzed by kinase enzymes is linked to a wide variety of
cancers. Similarly, the unregulated acetylation state of histone proteins, controlled by histone deacetylase
(HDAC) proteins, can lead to epigenetic changes in transcription and ultimately disease. Key to
characterizing both healthy and disease states is a detailed molecular understanding of the role of protein
post-translational modifications, such as phosphorylation and acetylation, on protein function and
interactions. Importantly, enzymes regulating post-translational modifications, including kinase and HDAC
proteins, are targets of drug treatment. Yet, tools linking protein modifications to downstream biological
activities are often limited or unavailable, which has stalled progress in disease characterization and drug
development.
The NIGMS-funded projects in the Pflum lab address the critical need to develop innovative chemical
approaches to discover unanticipated functions of protein modifying enzymes in cell biology. In our work
with protein phosphorylation, we have pioneered in the last 10 years application of ATP analogs in kinase-
catalyzed labeling reactions to develop a suite of methods to probe kinase-substrate pairs and multi-protein
complexes in cells. This supplemental funding application will support this project by applying our innovative
tools to complex biological problems in collaboration with biologists. In our work with protein acetylation, we
have demonstrated in the last several years the power of using trapping mutants to discover non-histone
substrates of HDAC1, which has revealed unexpected roles of HDAC1 proteins in cell biology. In this
supplemental funding application, we will apply this powerful trapping strategy to discover the substrates of
HDAC1 in sickle cell anemia model systems, which will reveal unanticipated molecular mechanisms in sickle
cell and possible new therapeutics. Importantly, these studies will provide an ideal training ground to expose
the pre-doctoral candidate to proteomics-based mass spectrometry, biochemistry, and cell biology to
prepare for entry into the biomedical workforce.

## Key facts

- **NIH application ID:** 10410612
- **Project number:** 3R35GM131821-03S1
- **Recipient organization:** WAYNE STATE UNIVERSITY
- **Principal Investigator:** Mary Kay H Pflum
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $56,384
- **Award type:** 3
- **Project period:** 2019-06-01 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10410612

## Citation

> US National Institutes of Health, RePORTER application 10410612, Chemical Approaches to Characterizing Protein Post-translational Modifications by Mass Spectrometry (3R35GM131821-03S1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10410612. Licensed CC0.

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