Inflect: Optimizing Computational Workflows for Thermal Proteome Profiling Data Analysis

The CETSA and Thermal
Proteome Profiling (TPP) analytical methods
are invaluable for the study of protein–ligand interactions
and protein stability in a cellular context. These tools have increasingly
been leveraged in work ranging from understanding signaling paradigms
to drug discovery. Consequently, there is an important need to optimize
the data analysis pipeline that is used to calculate protein melt
temperatures (Tm) and relative melt shifts
from proteomics abundance data. Here, we report a user-friendly analysis
of the melt shift calculation workflow where we describe the impact
of each individual calculation step on the final output list of stabilized
and destabilized proteins. This report also includes a description
of how key steps in the analysis workflow quantitatively impact the
list of stabilized/destabilized proteins from an experiment. We applied
our findings to develop a more optimized analysis workflow that illustrates
the dramatic sensitivity of chosen calculation steps on the final
list of reported proteins of interest in a study and have made the
R based program Inflect available for research community use through
the CRAN repository [McCracken, N. Inflect: Melt Curve Fitting and
Melt Shift Analysis. R package version 1.0.3, 2021].
The Inflect outputs include melt curves for each protein which passes
filtering criteria in addition to a data matrix which is directly
compatible with downstream packages such as UpsetR for replicate comparisons
and identification of biologically relevant changes. Overall, this
work provides an essential resource for scientists as they analyze
data from TPP and CETSA experiments and implement their own analysis
pipelines geared toward specific applications.