Comparative Analysis of Protein Folding Stability-Based Profiling Methods for Characterization of Biological Phenotypes

Recently, a new suite
of mass spectrometry-based proteomic methods
has been developed that enables evaluation of protein folding stability
on the proteomic scale. These methods utilize chemical and thermal
denaturation approaches (SPROX and TPP, respectively) as well as proteolysis
strategies (DARTS, LiP, and PP) to assess protein folding stability.
The analytical capabilities of these technique have been well-established
for protein target discovery applications. However, less is known
about the relative advantages and disadvantages of using these different
strategies to characterize biological phenotypes. Reported here is
a comparative study of SPROX, TPP, LiP, and conventional protein expression
level measurements using both a mouse model of aging and a mammalian
cell culture model of breast cancer. Analyses on proteins in brain
tissue cell lysates derived from 1- and 18-month-old mice (n = 4–5 at each time point) and on proteins in cell
lysates derived from the MCF-7 and MCF-10A cell lines revealed a majority
of the differentially stabilized protein hits in each phenotype analysis
had unchanged expression levels. In both phenotype analyses, TPP generated
the largest number and fraction of differentially stabilized protein
hits. Only a quarter of all the protein hits identified in each phenotype
analysis had a differential stability that was detected using multiple
techniques. This work also reports the first peptide-level analysis
of TPP data, which was required for the correct interpretation of
the phenotype analyses performed here. Studies on selected protein
stability hits also uncovered phenotype-related functional changes.