INDIANA UNIVERSITY SCHOOL OF MEDICINE
DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY
CENTER FOR COMPUTATIONAL BIOLOGY AND BIOINFORMATICS
NEWS
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September 2024 - Congratulations Tara on advancing to candidacy!
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May 2024 - Welcome Katelyn Caric to the Mosley lab!
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September 2023 - Congratulations to Dr. Mosley for being awarded the Outstanding Mentor Award from Biochemistry and Molecular Biology Students!
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@MosleyLab
OUR LATEST RESEARCH
Obtaining Functional Proteomics Insights From TPP Through Optimized Melt Shift Calculation and Statistical Analysis With InflectSSP
Thermal proteome profiling (TPP) is an invaluable tool for functional proteomics studies that has been shown to discover changes associated with protein–ligand, protein–protein, and protein–RNA interaction dynamics along with changes in protein stability. The gap between data acquisition and data analysis tools is important to fill as TPP findings have reported subtle melt shift changes related to signaling events such as protein posttranslational modifications. In this study, we have improved the Inflect data analysis pipeline (now referred to as InflectSSP) to increase the sensitivity of detection for both large and subtle changes in the proteome as measured by TPP. Specifically, InflectSSP now has integrated statistical and bioinformatic functions to improve objective functional proteomics findings from the quantitative results obtained from TPP studies through increasing both the sensitivity and specificity of the data analysis pipeline. InflectSSP incorporates calculation of a “melt coefficient” into the pipeline with production of average melt curves for biological replicate studies to aid in identification of proteins with significant melts.
Boosting Detection of Low Abundance Proteins in TPP Experiments
The study of low-abundance proteins is a challenge to discovery-based proteomics. Mass spectrometry (MS) applications, such as thermal proteome profiling (TPP), face specific challenges in the detection of the whole proteome as a consequence of the use of nondenaturing extraction buffers. We incorporated an affinity-purified protein complex sample at submolar concentrations as an isobaric trigger channel into a mutant TPP (mTPP) workflow to provide reproducible detection and quantitation of the low-abundance proteins. Overall, the incorporation of an affinity-purified protein complex as an isobaric trigger channel within a tandem mass tag (TMT) multiplex for mTPP experiments is an effective and reproducible way to gather thermal profiling data on proteins that are not readily detected using the original TPP or mTPP protocols.
Temperature sensitive Mutant Proteome Profiling (TeMPP)
Temperature sensitive Mutant Proteome Profiling (TeMPP) is a novel application of mass spectrometry (MS) based thermal proteome profiling (TPP) to characterize effects of missense mutations on protein stability and protein-protein interactions. This study characterizes missense mutations in two different subunits of the 26S proteasome on the thermal stability of the proteome at large, revealing distinct mechanistic details that were not obtained using only steady-state transcriptome and proteome analyses. Overall, TeMPP is a precise, unbiased approach to rapidly measure changes in missense mutant containing proteomes without the requirement for large amounts of starting material, specific antibodies against proteins of interest, and/or genetic manipulation of the biological system.
Dial-In on how Phosphatase Rtr1 Fine Tunes Transcription Termination
The C-terminal domain (CTD) of the largest subunit of RNAPII is dynamically phosphorylated during transcription. The CTD phosphorylation status is critical for the recruitment and interaction of transcription regulatory proteins with RNAPII. In this manuscript, we present data that shows that the CTD phosphatase Rtr1 restricts early termination of RNAPII transcription.
Six degrees of RNA Polymerase II—Interaction Network Building using Mass Spectrometry
RNA Polymerase II (RNAPII) is the protein complex responsible for transcribing mRNA and many non-coding RNA. Proper transcription of these RNA is critical to maintaining gene expression and cell health. Consequently, RNAPII interacts with a multitude of other proteins that aid in proper regulation of transcription. This article reviews how mass spectrometry has helped to tease out the intricate and dynamic protein-protein interaction network of RNAPII that is responsible for maintaining transcription.
Transcription-Coupled RNA Quality Control: A lesson in how to check yourself before you wreck yourself
Nascent RNA that are improperly processed or contain transcription errors pose a threat to cell health, and can even cause disease. Fortunately, RNAPII transcriptions are processed for quality control in tandem with transcription. Processing machinery is recruited to RNAPII at the site of transcription and this article reviews these known and emerging co-transcriptional quality control mechanisms.
