# Thermal proteome profiling for analysis of protein sequence variants in human genetic disease

> **NIH NIH R01** · INDIANA UNIVERSITY INDIANAPOLIS · 2022 · $386,387

## Abstract

Project Summary
Many global proteomics studies have focused on the measurement of protein abundance and post-
translational modification status as measures of cellular function. These approaches, while highly informative,
are not sufficient as standalone approaches to address the role of genetic variation on human protein function
in a high-throughput manner. Our preliminary findings show that thermal proteome profiling (TPP) can
measure changes in missense mutant protein stability as well as the impacts of mutant protein stability
changes on protein-protein interactions (PPIs). We hypothesize that TPP will be sufficient to provide molecular
characterization of protein biophysical changes as a consequence of missense mutations associated with
human genetic disease. The initial work will focus on the optimization of TPP dataset analysis through
development of normalization approaches, curve fitting, and determination of key quality control cutoffs for
applications related to human genetic diseases. In parallel, we will perform mutant TPP analysis of human
genetic disease-associated protein sequence variants in the RNA-DNA helicase Senataxin and in multiple
subunits of the human RNA exosome. Numerous mutations in Senataxin and subunits of the RNA exosome
have been clinically associated with the rare neurodegenerative diseases: Ataxia Oculomotor Apraxia 2
(AOA2), Amyotrophic Lateral Sclerosis 4 (ALS4), and PontoCerebellar Hypoplasia (PCH). In addition to these
clinically characterized mutations, a number of additional variants have been identified of unknown clinical
significance. We propose that mutant TPP could be used to determine if these uncharacterized sequence
variants have similar or unique thermal profiles relative to known disease- causing variants. In addition to
mutant TPP analyses, we will perform RNA-Sequencing and chromatin immunoprecipitation analysis of a
selection of mutants to further delve into the functional consequences of mutant protein expression. Changes
in gene dosage for disease causing mutants and the impact of gene dosage on TPP outcomes will also be
explored through our recently developed approach for allele-specific thermal profiling. Allele-specific thermal
profiling is not possible through non-mass spectrometry-based methods such as ELISA since determination of
protein sequence will be required to differentiate the slight changes in protein sequence. Long-term goals
include development of mutant TPP analysis to facilitate measurement of protein expression buffering of
deleterious alleles, protein-protein interaction network changes, and the impact of altered variant protein levels
on the correlation between mRNA and protein abundance levels.

## Key facts

- **NIH application ID:** 10349591
- **Project number:** 5R01NS121550-02
- **Recipient organization:** INDIANA UNIVERSITY INDIANAPOLIS
- **Principal Investigator:** AMBER L. MOSLEY
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $386,387
- **Award type:** 5
- **Project period:** 2021-02-15 → 2026-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10349591

## Citation

> US National Institutes of Health, RePORTER application 10349591, Thermal proteome profiling for analysis of protein sequence variants in human genetic disease (5R01NS121550-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10349591. Licensed CC0.

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