High throughput discovery of functional protein modifications by Hotspot Thermal Profiling

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Jun X Huang1,2, Gihoon Lee1,2, Kate E Cavanaugh3,4,5

  • 1Department of Chemistry, The University of Chicago, Chicago, IL, USA.

Nature Methods
|August 7, 2019
PubMed

Insights

This study introduces Hotspot Thermal Profiling, a new proteomic method to identify how protein phosphorylation affects protein stability and function in live cells, enabling unbiased discovery of critical modification sites.

Area of Science:

  • Proteomics
  • Biophysics
  • Cellular Biology

Background:

  • Mass spectrometry allows global analysis of post-translationally modified proteoforms.
  • Predicting or prioritizing modification sites that impact protein function remains a challenge.

Purpose of the Study:

  • To develop a method for detecting the impact of site-specific protein phosphorylation on protein thermal stability in live cells.
  • To systematically identify and prioritize functional 'hotspot' protein modification sites.

Main Methods:

  • Hotspot Thermal Profiling (HTP), a massively parallel biophysical assay.
  • Analysis of thermal stability shifts in thousands of native proteins in response to phosphorylation.
  • Detection of intrinsic structural changes and extrinsic interaction changes.

Main Results:

  • HTP detected site-specific phosphorylation effects on protein thermal stability.
  • Observed trends related to protein function and structure.
  • Demonstrated high-throughput, unbiased discovery and prioritization of functional modification sites.

Conclusions:

  • Hotspot Thermal Profiling is a powerful tool for studying post-translational modifications.
  • The method can reveal how phosphorylation impacts protein stability, structure, and interactions.
  • Applicable across diverse organisms, cell types, and modifications for functional site discovery.

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