Protocol to quantify protein phosphorylation in mouse extended pluripotent stem cell-derived blastoids

Protein phosphorylation is an important post-transcription modification regulating essential cellular activities. Here, we present a proteomics protocol to quantify protein phosphorylation in the extended pluripotent stem cell (EPSC)-derived blastoids from Mus musculus with a standardized phosphoproteome analysis workflow. We describe steps for culturing blastoids, preparing samples, enriching phosphopeptides, and fractionating peptides. We then detail procedures for acquiring and analyzing high-resolution liquid chromatography-mass spectrometry (LC-MS) data. This protocol provides a framework for reproducible analysis of phospho-proteomes of mouse blastoids. For complete details on the use and execution of this protocol, please refer to Min et al.¹.