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

Area of Science:

• Proteomics
• Biochemistry
• Systems Biology

Background:

• Mass spectrometry-based proteomic methods can now assess protein folding stability at a large scale.
• Techniques include chemical denaturation (SPROX), thermal denaturation (TPP), and proteolysis (DARTS, LiP, PP).
• While useful for target discovery, their comparative performance in characterizing biological phenotypes is less understood.

Purpose of the Study:

• To compare the utility of SPROX, TPP, and LiP, alongside traditional protein expression measurements, for characterizing biological phenotypes.
• To investigate protein stability changes in a mouse model of aging and a mammalian cell culture model of breast cancer.

Main Methods:

• Comparative analysis of SPROX, TPP, and LiP methods using mouse brain tissue from young and aged mice.
• Analysis of MCF-7 and MCF-10A breast cancer cell lines using the same proteomic techniques.
• Measurement of conventional protein expression levels for comparison.
• First-time peptide-level analysis of TPP data.

Main Results:

• A majority of differentially stabilized proteins identified in both aging and cancer models showed no significant change in expression levels.
• Thermal proteome profiling (TPP) identified the highest number and proportion of differentially stabilized protein hits in both models.
• Only about 25% of identified protein stability hits were detected by multiple techniques.
• Phenotype-related functional changes were uncovered in selected protein stability hits.

Conclusions:

• Protein folding stability, rather than expression level, is a key indicator of biological phenotypes such as aging and cancer.
• TPP is a powerful method for identifying phenotype-specific protein stability alterations.
• Multiple techniques are needed to comprehensively capture protein stability changes, as different methods detect distinct sets of alterations.