From In-Vitro Protein Assembly to In-Situ Functional Analysis: Cell-Permeable Synthetic Protein Probes for Exploring Ubiquitination and PTM Biology in Native Cellular Environments

Conspectus Post-translational modifications (PTMs) are mainly low-abundance, transient, and spatiotemporally regulated molecular events that govern protein function across nearly all cellular processes, including protein degradation, inflammatory signaling, DNA damage repair, and chromatin regulation. Recent advances in genetic code expansion and proximity labeling have substantially expanded the toolkit for studying PTM-mediated processes in live cells. However, these approaches typically rely on genetic manipulation, which limits their application in primary cells, hard-to-transfect cell lines, clinical specimens, and other native cell systems (genetically unmodified live cells) where minimal perturbation is essential. The continued development of chemical protein synthesis has made it possible to access structurally defined PTM-modified protein tools with extraordinary precision. Yet, the central challenge for their in-vivo application is no longer simply the preparation of defined proteoforms, but rather how to deliver these in-vitro-assembled protein tools into native cells in an active form and translate them into functionally interpretable biological signals. In this Account, we summarize a series of studies carried out in our laboratory since 2020, organized around the theme of “chemical synthesis in vitro ─ delivery into live cells ─ functional analysis in situ”. By combining solid-phase peptide synthesis, protein segment ligation, and enzymatic modification, we have introduced precisely defined PTMs, photo-cross-linking groups, and affinity handles into ubiquitin (Ub) and histone PTM probes. For relatively small and structurally stable tools (e.g., mono-Ub, di-Ub, and histone-tail probes), covalent conjugation with cell-penetrating peptides (CPPs) enables efficient delivery into native cells, allowing in-situ interrogation of Ub writers, erasers, and modification-dependent protein interaction networks. When the probe architecture expands to more-complex and conformationally sensitive species, such as E2–Ub conjugates, we developed a noncovalent CPP cluster assembly strategy to increase the local positive charge density of proteins and to minimize the interference of CPP conjugation with protein conformation and target recognition. This approach enabled the delivery of E2–Ub structure-mimetic probes into live cells and the mapping of stimulus-dependent E2–E3 interaction networks in situ. Through these case studies, we emphasize that the design of cell-permeable synthetic PTM probes should not only consider how to obtain structurally defined proteoforms, but also take into account compatible delivery strategies and intracellular function maintenance at the probe construction stage, so as to advance synthetic protein tools beyond “structurally precise assembly” toward “functional interrogation in native cells”.