Top-Down Thermal Proteome Profiling (TD-TPP) for Functional Characterization of the Intact Proteoforms in Complex...

Area of Science:

• Proteomics
• Biochemistry
• Molecular Biology

Background:

• Protein function is influenced by structural variations like post-translational modifications (PTMs) and amino acid changes.
• Functional proteomics aims to understand protein function alterations within biological pathways.
• Existing thermal proteome profiling (TPP) methods use bottom-up approaches, requiring protein digestion that can lose intact proteoform structural data.

Purpose of the Study:

• To develop a novel top-down (TD) TPP approach for analyzing intact proteoform thermal stability.
• To create a high-throughput data analysis pipeline for label-free quantitative analysis and identification.
• To assess the TD-TPP platform's capability in detecting thermal stability shifts due to genetic variations, ligand binding, and in complex biological samples.

Main Methods:

• Development of a top-down (TD) TPP platform for intact proteoform stability analysis.
• Implementation of a high-throughput, label-free quantitative analysis and identification pipeline.
• Benchmarking using standard proteins (β-lactoglobulin A/B) and ligand-protein interactions (carbonic anhydrase/acetazolamide).
• Application to high-throughput thermal stability profiling of the intact *E. coli* proteome.

Main Results:

• The TD-TPP platform successfully differentiated thermal stability between β-lactoglobulin A and B, attributed to amino acid substitutions.
• The platform detected thermal stabilization of carbonic anhydrase upon binding with acetazolamide.
• High-throughput profiling of *E. coli* lysate identified and characterized 163 proteoforms, with melting points correlating well with bottom-up TPP methods.

Conclusions:

• The developed TD-TPP platform enables the study of intact proteoform thermal stability.
• This method is effective for detecting thermal stability changes induced by genetic variations and ligand binding.
• The TD-TPP platform is suitable for high-throughput thermal stability profiling in complex biological samples, providing proteoform-level insights.