INDIANA UNIVERSITY SCHOOL OF MEDICINE
DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY
CENTER FOR COMPUTATIONAL BIOLOGY AND BIOINFORMATICS
PROJECTS
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Measuring Systems Level Effects of Rare Genetic
Disease Sequence Variants using Quantitative Proteomics
1. Images of neurodegeneration specific to the pons and cerebellum seen in Pontocerebellar hypoplasia 1b.
2. Genome sequencing can identify potential rare disease variants, but cannot characterize protein-level functional changes.
3. Protein sequence variants can lead to a wide array of functional changes which require protein-level quantitative measurement for assessment.
4. We are developing new computational & quantitative measurement methods in custom genetic model systems.
5. Includes thermal proteome profiling (TPP) - a biophysical approach that can identify protein functional changes including drug-target interactions.
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Omics based Analysis of RPAP2 function within Transcription and the Unfolded Protein Response
1. Yeast RPAP2/ Human RPAP2 is a protein phosphatase that regulates transcription by dephosphorylating RNA Polymerase II
2. RPAP2 has recently been observed within the regulation of the unfolded protein response by dephosphorylating the unfolded protein sensor IRE1 to allow for the signaling cascade from an additional unfolded protein sensor PERK to progress.
We are currently conducting a host of Omics-based analysis to further characterize RPAP2 function.
Drug-based Interrogation Phosphorylation Changes within Transcription Elongation using FAIMS
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1. Within Mass spectrometry (MS)-based quantitative proteomics analysis, there is an ongoing goal to optimize reproducible identification and quantitation of peptides while still allowing for discovery-based workflows.
2. High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) allows for a gas phase chromatography step to be inserted into a general MS workflow that assists with the separation of peptides ions before reaching the MS detector.
3. Within Transcription Elongation, promotor proximal pausing occurs to maintain mRNA generation fidelity by stalling RNA polymerase II on target DNA regions. CDK9 phosphorylates RNA polymerase II as well as other regulatory factors to allow for processive transcription elongation.
We are currently further characterizing transcription elongation through the use of phosphoproteomics workflows that include the utilization of FAIMS and inhibition of transcription elongation by CDK9 inhibitor flavopiridol.