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Updated: Jan 21, 2026

High-Throughput Assays of Critical Thermal Limits in Insects
Published on: June 15, 2020
High throughput discovery of functional protein modifications by Hotspot Thermal Profiling
Jun X Huang1,2, Gihoon Lee1,2, Kate E Cavanaugh3,4,5
1Department of Chemistry, The University of Chicago, Chicago, IL, USA.
Abstract:
Mass spectrometry enables global analysis of posttranslationally modified proteoforms from biological samples, yet we still lack methods to systematically predict, or even prioritize, which modification sites may perturb protein function. Here we describe a proteomic method, Hotspot Thermal Profiling, to detect the effects of site-specific protein phosphorylation on the thermal stability of thousands of native proteins in live cells. This massively parallel biophysical assay unveiled shifts in overall protein stability in response to site-specific phosphorylation sites, as well as trends related to protein function and structure. This method can detect intrinsic changes to protein structure as well as extrinsic changes to protein-protein and protein-metabolite interactions resulting from phosphorylation. Finally, we show that functional 'hotspot' protein modification sites can be discovered and prioritized for study in a high-throughput and unbiased fashion. This approach is applicable to diverse organisms, cell types and posttranslational modifications.
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