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

Protein function can vary due to changes in primary structure, such as post-translational modification (PTM), truncation, or amino acid replacement, and functional proteomics methods focus on elucidating changes in the function of proteins in biological pathways. Thermal proteome profiling (TPP) is a powerful functional proteomics approach that analyzes the thermal stability of proteins by exposing them to a temperature gradient to facilitate thermal denaturation, followed by analysis of the remaining folded proteins. Current TPP methods, however, utilize bottom-up methods that require protein digestion and can obscure relevant information regarding the structure of the intact proteoform. In this study, we have developed a top-down (TD) TPP approach to study intact proteoform stability as well as a high-throughput data analysis pipeline for label-free quantitative analysis and identification. We benchmarked this platform using two proteoforms of standard proteins, β-lactoglobulin A and β-lactoglobulin B (βLG-A and βLG-B), and found that βLG-A is slightly stabilized compared with βLG-B as a result of two amino acid substitutions. Additionally, we utilized this platform to detect protein thermal stability shifts induced by ligand binding, using carbonic anhydrase and its known inhibitor, acetazolamide. Our results demonstrated that the TD-TPP platform effectively detected the stabilization of a standard protein upon ligand binding. Furthermore, we adapted the TD-TPP platform for high-throughput thermal stability profiling of the intact E. coli proteome, enabling the characterization of intact proteoform-level thermal stability in complex biological samples. We performed thermal profiling of 72 identified proteoforms and 91 proteoform features (e.g., 163 total proteoforms) from intact E. coli lysate, and found that the melting points of these proteoforms correlated well with melting points determined using bottom-up TPP methods. Overall, the TD-TPP platform is capable of profiling thermal stability for standard proteins and intact proteoforms in complex biological samples.