Developing a Novel and Integrative Method to Systematically Investigate the Stability of N-Terminal Proteoforms

Protein N-terminal proteoforms, which include both N-terminal modifications and sequence variation arising from processes such as proteolytic cleavage, are common in cells and have been reported to significantly impact protein stability. Systematically examining the correlation between these N-terminal proteoforms and protein stability is essential for understanding their biological roles. Recently, thermal stability analysis has been applied to study protein modifications. Here, we combined Thermal Proteome Profiling (TPP) with tandem mass tag (TMT)-Terminal Amine Isotopic Labeling of Substrates (TAILS) to globally investigate the thermal stability of N-terminal proteoforms in THP-1 cells. The thermal stability of more than 13,000 unique N-terminal peptides corresponding to approximately 4,700 proteins were quantified. Overall, N-terminal proteoforms exhibited higher thermal stability than proteins identified in whole proteome group. A series of factors such as hydrophobicity and secondary structure influenced the thermal stability differences of N-terminal proteoforms.