Browsing by Subject "Protein stability"

Now showing 1 - 4 of 4
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    Boosting Detection of Low-Abundance Proteins in Thermal Proteome Profiling Experiments by Addition of an Isobaric Trigger Channel to TMT Multiplexes
    (American Chemical Society, 2021-05-11) Peck Justice, Sarah A.; McCracken, Neil A.; Victorino, José F.; Qi, Guihong D.; Wijeratne, Aruna B.; Mosley, Amber L.; Biochemistry and Molecular Biology, School of Medicine
    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. TPP is a powerful method for the study of protein thermal stability, but quantitative accuracy is highly dependent on consistent detection. Therefore, TPP can be limited in its amenability to study low-abundance proteins that tend to have stochastic or poor detection by MS. To address this challenge, 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 subunits of the cleavage and polyadenylation factor (CPF) complex. The inclusion of an isobaric protein complex trigger channel increased detection an average of 40× for previously detected subunits and facilitated detection of CPF subunits that were previously below the limit of detection. Importantly, these gains in CPF detection did not cause large changes in melt temperature (Tm) calculations for other unrelated proteins in the samples, with a high positive correlation between Tm estimates in samples with and without isobaric trigger channel addition. 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.
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    Genetic mosaicism, intrafamilial phenotypic heterogeneity, and molecular defects of a novel missense SLC6A1 mutation associated with epilepsy and ADHD
    (Elsevier, 2021) Poliquin, Sarah; Hughes, Inna; Shen, Wangzhen; Mermer, Felicia; Wang, Juexin; Mack, Taralynn; Xu, Dong; Kang, Jing-Qiong; Medical and Molecular Genetics, School of Medicine
    Background: Mutations in SLC6A1, encoding γ-aminobutyric acid (GABA) transporter 1 (GAT-1), have been recently associated with a spectrum of neurodevelopmental disorders ranging from variable epilepsy syndromes, intellectual disability (ID), autism and others. To date, most identified mutations are de novo. We here report a pedigree of two siblings associated with myoclonic astatic epilepsy, attention deficit hyperactivity disorder (ADHD), and ID. Methods: Next-generation sequencing identified a missense mutation in the SLC6A1 gene (c.373G > A(p.Val125Met)) in the sisters but not in their shared mother who is also asymptomatic, suggesting gonadal mosaicism. We have thoroughly characterized the clinical phenotypes: EEG recordings identified features for absence seizures and prominent bursts of occipital intermittent rhythmic delta activity (OIRDA). The molecular pathophysiology underlying the clinical phenotypes was assessed using a multidisciplinary approach including machine learning, confocal microscopy, and high-throughput 3H radio-labeled GABA uptake assays in mouse astrocytes and neurons. Results: The GAT-1(Val125Met) mutation destabilizes the global protein conformation and reduces transporter protein expression at total and cell surface. The mutant transporter protein was localized intracellularly inside the endoplasmic reticulum (ER) in both HEK293T cells and astrocytes which may directly contribute to seizures in patients. Radioactive 3H-labeled GABA uptake assay indicated the mutation reduced the function of the mutant GAT-1(Val125Met) to ~30% of the wildtype. Conclusions: The seizure phenotypes, ADHD, and impaired cognition are likely caused by a partial loss-of-function of GAT-1 due to protein destabilization resulting from the mutation. Reduced GAT-1 function in astrocytes and neurons may consequently alter brain network activities such as increased seizures and reduced attention.
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    Inflect: Optimizing Computational Workflows for Thermal Proteome Profiling Data Analysis
    (American Chemical Society, 2021-04-02) McCracken, Neil A.; Peck Justice, Sarah A.; Wijeratne, Aruna B.; Mosley, Amber L.; Biochemistry and Molecular Biology, School of Medicine
    The CETSA and Thermal Proteome Profiling (TPP) analytical methods are invaluable for the study of protein–ligand interactions and protein stability in a cellular context. These tools have increasingly been leveraged in work ranging from understanding signaling paradigms to drug discovery. Consequently, there is an important need to optimize the data analysis pipeline that is used to calculate protein melt temperatures (Tm) and relative melt shifts from proteomics abundance data. Here, we report a user-friendly analysis of the melt shift calculation workflow where we describe the impact of each individual calculation step on the final output list of stabilized and destabilized proteins. This report also includes a description of how key steps in the analysis workflow quantitatively impact the list of stabilized/destabilized proteins from an experiment. We applied our findings to develop a more optimized analysis workflow that illustrates the dramatic sensitivity of chosen calculation steps on the final list of reported proteins of interest in a study and have made the R based program Inflect available for research community use through the CRAN repository [McCracken, N. Inflect: Melt Curve Fitting and Melt Shift Analysis. R package version 1.0.3, 2021]. The Inflect outputs include melt curves for each protein which passes filtering criteria in addition to a data matrix which is directly compatible with downstream packages such as UpsetR for replicate comparisons and identification of biologically relevant changes. Overall, this work provides an essential resource for scientists as they analyze data from TPP and CETSA experiments and implement their own analysis pipelines geared toward specific applications.
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    Mutant thermal proteome profiling for characterization of missense protein variants and their associated phenotypes within the proteome
    (Elsevier, 2020-11-27) Peck Justice, Sarah A.; Barron, Monica P.; Qi, Guihong D.; Wijeratne, H.R. Sagara; Victorino, José F.; Simpson, Ed R.; Vilseck, Jonah Z.; Wijeratne, Aruna B.; Mosley, Amber L.; Biochemistry and Molecular Biology, School of Medicine
    Temperature-sensitive (TS) missense mutants have been foundational for characterization of essential gene function. However, an unbiased approach for analysis of biochemical and biophysical changes in TS missense mutants within the context of their functional proteomes is lacking. We applied MS-based thermal proteome profiling (TPP) to investigate the proteome-wide effects of missense mutations in an application that we refer to as mutant thermal proteome profiling (mTPP). This study characterized global impacts of temperature sensitivity-inducing missense mutations in two different subunits of the 26S proteasome. The majority of alterations identified by RNA-Seq and global proteomics were similar between the mutants, which could suggest that a similar functional disruption is occurring in both missense variants. Results from mTPP, however, provide unique insights into the mechanisms that contribute to the TS phenotype in each mutant, revealing distinct changes that were not obtained using only steady-state transcriptome and proteome analyses. Computationally, multisite λ-dynamics simulations add clear support for mTPP experimental findings. This work shows that mTPP is a precise approach to 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. Although experiments were performed under permissive conditions, mTPP provided insights into the underlying protein stability changes that cause dramatic cellular phenotypes observed at nonpermissive temperatures. Overall, mTPP provides unique mechanistic insights into missense mutation dysfunction and connection of genotype to phenotype in a rapid, nonbiased fashion.