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

## TL;DR

A new in-cell proteomics method allows detailed mapping of protein changes in early frog embryos, revealing key developmental shifts.

## Contribution

The novel in-cell proteomics strategy enables high-resolution, low-input proteomic analysis of early embryos without lysis or yolk depletion.

## Key findings

- A drastic proteomic shift was observed between 2- and 4-cell stages of embryo development.
- Spatial analysis showed strong molecular asymmetry along the animal–vegetal axis but minimal dorsal–ventral differences in 8-cell blastomeres.
- The method consistently quantified approximately 6200 proteins from a single X. tropicalis embryo.

## Abstract

Early embryonic development requires tightly regulated molecular programs to coordinate cell division, fate specification, and spatial patterning. While transcriptomic profiling has been widely performed, proteomic analyses of early vertebrate embryos remain limited owing to technical challenges in embryonic sample preparation. Here, we present an “in-cell proteomics” strategy, which bypasses cell lysis and yolk depletion, processes individual embryos directly in functionalized filter devices, and generates mass spectrometry (MS)-friendly samples in an extremely robust and streamlined manner. This single-vessel approach minimizes sample loss and technical variation, offering a highly sensitive and accurate alternative to low-input and low-cell quantitative proteomics. Coupled with field asymmetric ion mobility spectrometry and single-shot data-independent acquisition MS workflow, this approach enabled us to consistently quantify ∼6200 proteins from a single Xenopus tropicalis embryo, representing the deepest proteomic coverage of early X. tropicalis developmental stages reported to date. Investigation of the temporal proteomes across five cleavage stages (from 1- to 16-cell stages) 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, whereas dorsal–ventral differences were minimal. This study establishes a novel in-cell proteomics technology in conjunction with field asymmetric ion mobility spectrometry and data-independent acquisition 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.

•In-cell proteomics approach enables direct processing of proteins in the embryos.•The approach offers high sensitivity and low variability for embryonic proteomics.•Drastic proteome shift was revealed between 2- and 4-cell cleavage stages.•Dissection of 8-cell blastomeres uncovered minimal dorsal–ventral differences.

In-cell proteomics approach enables direct processing of proteins in the embryos.

The approach offers high sensitivity and low variability for embryonic proteomics.

Drastic proteome shift was revealed between 2- and 4-cell cleavage stages.

Dissection of 8-cell blastomeres uncovered minimal dorsal–ventral differences.

Conventional shotgun proteomic analysis of Xenopus embryos has been challenging due to tedious sample preparations, significant sample loss, and large variations. In this study, we developed an in-cell proteomics approach that can directly process proteins in the embryonic cells without lysis and extraction, offering the simplest approach reported to date for embryonic proteomics analysis. Applied to single Xenopustropicalis embryos and blastomeres, the approach showcased great potential for studying proteome-wide dynamics of early cleavage stages and dissecting dorsal–ventral differences.