Zebrafish is a well-recognized organism for investigating vertebrate development and human diseases. However, the data on zebrafish proteome are scarce, particularly during embryogenesis. This is mostly due to the overwhelming abundance of egg yolk proteins, which tend to mask the detectable presence of less abundant proteins. We developed an efficient procedure to reduce the amount of yolk in zebrafish early embryos to improve the Liquid chromatography–tandem mass spectrometry (LC–MS)-based shotgun proteomics analysis. We demonstrated that the deyolking procedure resulted in a greater number of proteins being identified. This protocol resulted in approximately 2-fold increase in the number of proteins identified in deyolked samples at cleavage stages, and the number of identified proteins increased greatly by 3–4 times compared to non-deyolked samples in both oblong and bud stages. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed a high number of functional proteins differentially accumulated in the deyolked versus non-deyolked samples. The most prominent enrichments after the deyolking procedure included processes, functions, and components related to cellular organization, cell cycle, control of replication and translation, and mitochondrial functions. This deyolking procedure improves both qualitative and quantitative proteome analyses and provides an innovative tool in molecular embryogenesis of polylecithal animals, such as fish, amphibians, reptiles, or birds.
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