Obtaining Functional Proteomics Insights From Thermal Proteome Profiling Through Optimized Melt Shift Calculation and...

Area of Science:

• Proteomics
• Biochemistry
• Computational Biology

Background:

• Thermal proteome profiling (TPP) is crucial for studying protein interactions and stability changes.
• Existing TPP data analysis methods lack sensitivity for subtle cellular signaling events.
• There is a need for improved analytical tools to fully leverage TPP data quality and sensitivity.

Purpose of the Study:

• To enhance the Inflect data analysis pipeline, now InflectSSP, for improved TPP data analysis.
• To increase the sensitivity and specificity of detecting proteome-wide changes from TPP experiments.
• To provide a robust R-based program for objective functional proteomics findings.

Main Methods:

• Integrated statistical and bioinformatic functions into the InflectSSP R package.
• Incorporated calculation of a 'melt coefficient' and average melt curves for biological replicates.
• Reanalyzed two previously reported TPP datasets and conducted a new study on thapsigargin-induced unfolded protein response.

Main Results:

• InflectSSP demonstrated improved sensitivity and specificity in detecting proteome changes from TPP data.
• Reanalysis of existing datasets validated the performance of the InflectSSP R package.
• Analysis of the unfolded protein response study revealed reproducible, significant target engagement and new mechanistic insights.

Conclusions:

• InflectSSP significantly advances TPP data analysis, enabling more sensitive and specific detection of proteome dynamics.
• The enhanced pipeline aids in identifying subtle melt shifts related to cellular signaling and posttranslational modifications.
• InflectSSP provides valuable insights into drug mechanisms of action, as demonstrated in the thapsigargin study.