Obtaining Functional Proteomics Insights From Thermal Proteome Profiling Through Optimized Melt Shift Calculation and Statistical Analysis With InflectSSP

McCracken, Neil A.; Liu, Hao; Runnebohm, Avery M.; Wijeratne, H. R. Sagara; Wijeratne, Aruna B.; Staschke, Kirk A.; Mosley, Amber L.

doi:10.1016/j.mcpro.2023.100630

Cited by 4 publications

(5 citation statements)

References 51 publications

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“…This involves pooling the soluble fraction from different temperature points and comparing global protein abundance between conditions rather than assessing the melting point 15 (Figure 1B). Others focus on refining downstream data analysis strategies for melting point determination 16 . We analyzed three different TPP studies and found that the median melting point of all proteins across experiments conducted in cells and cellular extracts typically falls between 50-55°C, with nearly 50% of all protein melting temperatures falling within this range (Supplementary Figure S1, Supplementary Table 1).…”

Section: Resultsmentioning

confidence: 99%

Streamlined Proteome-Wide Identification of Drug Targets Indicates Organ-Specific Engagement

Batth,

Locard-Paulet,

Doncheva

et al. 2024

Preprint

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Proteins are the primary targets of almost all small molecule drugs. However, even the most selectively designed drugs can potentially target several unknown proteins. Identification of potential drug targets can facilitate design of new drugs and repurposing of existing ones. Current state-of-the-art proteomics methodologies enable screening of thousands of proteins against a limited number of drug molecules. Here we report the development of a label-free quantitative proteomics approach that enables proteome-wide screening of small organic molecules in a scalable, reproducible, and rapid manner by streamlining the proteome integral solubility alteration (PISA) assay. We used rat organs ex-vivo to determine organ specific targets of medical drugs and enzyme inhibitors to identify novel drug targets for common drugs such as Ibuprofen. Finally, global drug profiling revealed overarching trends of how small molecules affect the proteome through either direct or indirect protein interactions.

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Section: Resultsmentioning

confidence: 99%

Streamlined Proteome-Wide Identification of Drug Targets Indicates Organ-Specific Engagement

Batth,

Locard-Paulet,

Doncheva

et al. 2024

Preprint

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“…Proteome-wide changes to protein-protein interactions, post-translational modifications, folding, and other biophysical properties of proteins can be assessed using thermal proteome profiling (TPP) (39–43). TPP is used to determine thermal stability of each protein under different conditions, which are then analyzed computationally to identify proteins and mechanisms impacted by treatment (44,45). Analysis of TPP data with the R-package InflectSSP produces melt curves and calculates melt temperatures (T m ) and significant shifts in protein melt temperature.…”

Section: Resultsmentioning

confidence: 99%

“…Analysis of TPP data with the R-package InflectSSP produces melt curves and calculates melt temperatures (T m ) and significant shifts in protein melt temperature. (45). TPP has been widely used to determine drug-target engagement (43), therefore we can apply it to test and compare the proteome-wide impacts on protein thermal stability across the different drug treatments.…”

Section: Resultsmentioning

confidence: 99%

IB-DNQ and Rucaparib dual treatment alters cell cycle regulation and DNA repair in triple negative breast cancer cells

Runnebohm,

Wijeratne,

Peck Justice

et al. 2024

Preprint

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Background: Triple negative breast cancer (TNBC), characterized by the lack of three canonical receptors, is unresponsive to commonly used hormonal therapies. One potential TNBC-specific therapeutic target is NQO1, as it is highly expressed in many TNBC patients and lowly expressed in non-cancer tissues. DNA damage induced by NQO1 bioactivatable drugs in combination with Rucaparib-mediated inhibition of PARP1-dependent DNA repair synergistically induces cell death. Methods: To gain a better understanding of the mechanisms behind this synergistic effect, we used global proteomics, phosphoproteomics, and thermal proteome profiling to analyze changes in protein abundance, phosphorylation and protein thermal stability. Results: Very few protein abundance changes resulted from single or dual agent treatment; however, protein phosphorylation and thermal stability were impacted. Histone H2AX was among several proteins identified to have increased phosphorylation when cells were treated with the combination of IB-DNQ and Rucaparib, validating that the drugs induced persistent DNA damage. Thermal proteome profiling revealed destabilization of H2AX following combination treatment, potentially a result of the increase in phosphorylation. Kinase substrate enrichment analysis predicted altered activity for kinases involved in DNA repair and cell cycle following dual agent treatment. Further biophysical analysis of these two processes revealed alterations in SWI/SNF complex association and tubulin / p53 interactions. Conclusions: Our findings that the drugs target DNA repair and cell cycle regulation, canonical cancer treatment targets, in a way that is dependent on increased expression of a protein selectively found to be upregulated in cancers without impacting protein abundance illustrate that multi-omics methodologies are important to gain a deeper understanding of the mechanisms behind treatment induced cancer cell death.

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“…The copyright holder for this preprint this version posted December 17, 2023. ; https://doi.org/10.1101/2023.12.17.571949 doi: bioRxiv preprint Reporter ion isotope interference was observed in empty TMT channels when using a meSIILCC at 10-fold the total protein of the lowest heated MS-TSA Sigmoidal curve fitting of normalized reporter ion intensities is a common approach to determine melting temperatures (Tm) for MS-TSA. 13,36,37 However, isotope impurities within isobaric labeling reagents can be problematic when employing SIILCC. [38][39][40][41] We assessed isotopic interference from meSIILCC by conducting triplicate MS-TSA experiments using only dimethyl sulfoxide (DMSO)-treated samples with and without meSIILCC.…”

Section: A Commercially Available Mp Enrichment Kit Was Selected To E...mentioning

confidence: 99%

“…To a lesser extent, the 130C, 131N, 133N, and 134C channels also show much lower levels of isotope contamination, which could be addressed by the implementation of correction factors. 13,36 The copyright holder for this preprint this version posted December 17, 2023. ; https://doi.org/10.1101/2023.12.17.571949 doi: bioRxiv preprint…”

Section: A Commercially Available Mp Enrichment Kit Was Selected To E...mentioning

confidence: 99%

Implementation of an enriched membrane protein carrier channel for enhanced detection of membrane proteins in mass spectrometry-based thermal stability assays

Phaneuf,

Ivanov

2023

Preprint

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In this work, we developed a membrane-enriched stable isotope isobaric-labeled carrier channel (meSIILCC) for mass spectrometry-based thermal stability assay (MS-TSA). A proof-of-concept study demonstrated that the meSIILCC method could modestly improve membrane protein (MP) detection in MS-TSA experiments. An enhancement of 10% in identifications of membrane-proteins was observed in the meSIILCC group. Hydrophobicity analysis of the identified and quantified peptides using the grand average of hydropathy index confirmed the meSIILCC approach enriched for peptides of higher hydrophobicity characteristic of membrane-associated proteins.To further improve meSIILCC, four membrane-protein enrichment approaches were compared. Using the selected and optimized workflow that utilized isobaric labeling-mass spectrometry, 8,662 protein groups were quantitatively characterized and then annotated based on their subcellular localization. The corresponding reporter ion intensities were used to construct a heatmap, which revealed an increased representation of proteins corresponding to the “plasma membrane” gene ontology term.In a separate DMSO-only MS-TSA experiment, the optimally performing meSIILCC was added at 10-fold the protein content of the lowest heated aliquot from the MS-TSA, and isotope interference was found to be the highest in the 134N channel, while to a much lesser degree in other channels that were left empty.To further assess the performance of meSIILCC in the DMSO-only MS-TSA experiment, an over-representation analysis was performed, which demonstrated that proteins exclusive to the meSIILCC group had more than a five-fold increase in gene ontology cellular component terms related to the “membrane” term.We found 496 proteins from the DMSO-only MS-TSA experiment, which were identified across all replicates and shared between the meSIILCC and control that were annotated with “plasma membrane.” A close to 28% increase in the set corresponding to unique peptides was realized, using the meSIILCC approach, with a median value of 6.3 peptides per protein, compared to 4.7 in the control.

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Obtaining Functional Proteomics Insights From Thermal Proteome Profiling Through Optimized Melt Shift Calculation and Statistical Analysis With InflectSSP

Cited by 4 publications

References 51 publications

Streamlined Proteome-Wide Identification of Drug Targets Indicates Organ-Specific Engagement

Streamlined Proteome-Wide Identification of Drug Targets Indicates Organ-Specific Engagement

IB-DNQ and Rucaparib dual treatment alters cell cycle regulation and DNA repair in triple negative breast cancer cells

Implementation of an enriched membrane protein carrier channel for enhanced detection of membrane proteins in mass spectrometry-based thermal stability assays

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