In-cell Proteomics Enables High-Resolution Spatial and Temporal Mapping of Early Xenopus tropicalis Embryos

Early embryonic development requires tightly regulated molecular programs to coordinate cell division, fate specification, and spatial patterning. While transcriptomic profiling is widely performed, proteomic analyses of early vertebrate embryos remain limited due to technical challenges in embryonic sample preparation. Here, we propose an “in-cell proteomics” approach, which bypasses cell lysis and yolk depletion, processes individual embryos directly in functionalized filter devices, and generates liquid chromatography–mass spectrometry (LC-MS)-friendly samples in an extremely robust and streamlined manner. Combined with a single-shot data-independent acquisition (DIA) MS workflow, this approach enabled us to consistently quantify ∼6,200 proteins from a single Xenopus tropicalis embryo, representing the deepest proteomic coverage of early X. tropicalis development reported to date. Investigation of the temporal proteomes across five cleavage stages (1- to 16-cell) revealed a drastic proteomic shift between 2- and 4-cell stages, followed by more gradual transitions thereafter. Spatial analysis of dissected 8-cell blastomeres uncovered pronounced molecular asymmetry along the animal–vegetal axis, while dorsal–ventral differences were minimal. This study establishes a novel in-cell proteomics technology in conjunction with DIA-MS as a robust platform for high-resolution, low-input developmental proteomics analysis, and provides a comprehensive spatiotemporal protein atlas for early X. tropicalis embryos.