# Administrative Supplement to Characterizing proteasome-substrate interactions by mass spectrometry proteomics

> **NIH NIH R01** · TRUSTEES OF INDIANA UNIVERSITY · 2021 · $50,000

## Abstract

PROJECT SUMMARY
 We are proposing to purchase a Thermo FAIMS Pro Orbitrap Ion Source that will attach to the front
of a Lumos Orbitrap and be used in our ubiquitin-proteasome cross-linking experiments. Our research
project involves probing interactions between polyubiquitin chains, model proteasome substrates and
the yeast proteasome. Cross-linking mass spectrometry is being applied to discover novel ubiquitin
receptors and to better understand how tagged substrates can impact proteasome function. It is crucial
that cross-links are identified with high confidence because of the complexity of the biochemical assays
that build on these results. Since there is limited consensus in this field as to what constitutes an
unambiguous cross-link identification, we are exerting considerable effort to improve cross-linking
observation and interpretation methodology. By combining electron transfer dissociation, collision
induced dissociation, and high energy collision induced dissociation of precursor ions we have recently
reported how the credibility of cross-link identifications can be enhanced. Crucial to the success of this
approach is to have intense precursor ion signals. The Thermo FAIMS Pro Orbitrap Ion Source is
designed to increase the transmission of highly-charged ions such as those associated with cross-
linked peptides and to discriminate against background species such as individual peptides. In fact, it
has already demonstrated success in cross-linking mass spectrometry experiments. We will use this
device to discover and confirm cross-links involving polyubiquitin and the yeast proteasome and by
doing so will achieve both analytical and biological advances. Progress in our ability to recognize and
conclusively identify cross-linked peptides has transformative potential for elucidating some of the most
important and challenging issues in protein science. Specifically, our cross-linking proteomics
experiments will provide candidates for novel substrate receptors on the proteasome and will identify
the sizes and linkage patterns in polyubiquitin that are most effective for recognition by the proteasome.
In summary, this project should result in significant advances in cross-linking methodology and in our
understanding of how the proteasome recognizes and digests substrates.

## Key facts

- **NIH application ID:** 10388694
- **Project number:** 3R01GM135264-02S1
- **Recipient organization:** TRUSTEES OF INDIANA UNIVERSITY
- **Principal Investigator:** DAVID E. CLEMMER
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $50,000
- **Award type:** 3
- **Project period:** 2020-07-01 → 2025-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10388694

## Citation

> US National Institutes of Health, RePORTER application 10388694, Administrative Supplement to Characterizing proteasome-substrate interactions by mass spectrometry proteomics (3R01GM135264-02S1). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10388694. Licensed CC0.

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