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.

Automated summary

A new suite of mass spectrometry-based proteomic methods has been developed for evaluating protein folding stability, using chemical and thermal denaturation approaches as well as proteolysis strategies. This study compared the advantages and disadvantages of using different techniques to characterize biological phenotypes. The results showed that TPP generated the largest number of differentially stabilized protein hits, with only a quarter of the hits detected by multiple techniques.