Proteomics

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Selection of Heating Temperatures Improves the Sensitivity of Proteome Integral Solubility Alteration (PISA) Assay


ABSTRACT: The thermal shift assay is a robust method of discovering protein-ligand interactions by measuring the alterations in protein thermal stability under various conditions. Several thermal shift assays have been developed and their throughput has been advanced greatly by the rapid progress in tandem mass tag-based quantitative proteomics. A recent paper by Gaetani et al. (J Proteome Res 2019, 18 (11), 4027-4037) introduced proteome integral solubility alteration (PISA) assay, further increasing throughput and simplifying the data analysis. Yet, it remains unclear how fold changes (integral treated samples versus integral control samples) perform in this assay. We show that fold changes have compromised linearity with ΔTm (shift in melting points) by simulation, and the magnitudes of the fold changes are inherently small in PISA assay, which is a challenge for quantitation. Both simulation and experimental results show that the selection of heating temperatures can tackle the small fold change problem and improve the sensitivity and specificity of the PISA assay.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Jiaming Li  

LAB HEAD: Steven P Gygi

PROVIDER: PXD017346 | Pride | 2020-04-06

REPOSITORIES: Pride

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Selection of Heating Temperatures Improves the Sensitivity of the Proteome Integral Solubility Alteration Assay.

Li Jiaming J   Van Vranken Jonathan G JG   Paulo Joao A JA   Huttlin Edward L EL   Gygi Steven P SP  

Journal of proteome research 20200413 5


The thermal shift assay is a robust method of discovering protein-ligand interactions by measuring the alterations in protein thermal stability under various conditions. Several thermal shift assays have been developed and their throughput has been advanced greatly by the rapid progress in tandem mass tag-based quantitative proteomics. A recent paper by Gaetani et al. ( <i>J. Proteome Res.</i> 2019, 18 (11), 4027-4037) introduced the proteome integral solubility alteration (PISA) assay, further  ...[more]

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