Rapid and High-Sensitive Phosphoproteomics Elucidated the Spatial Dynamics of the Mouse Brain

Recent developments in phosphoproteomics have enabled signaling studies where over 10,000 phosphosites can be routinely identified and quantified. Yet, current analyses are limited in sample size, reproducibility, and robustness, hampering experiments that involve low-input samples such as rare cells and fine-needle aspiration biopsies. To address these challenges, we introduced a simple and rapid phosphorylation enrichment method (miniPhos) that uses a minimal amount of the sample to get enough information to decipher biological significance. The miniPhos approach completed the sample pretreatment within 4 h and high effectively collected the phosphopeptides in a single-enrichment format with an optimized enrichment process and miniaturized system. This resulted in an average of 22,000 phosphorylation peptides quantified from 100 μg of proteins and even confidently localized over 4500 phosphosites from as little as 10 μg of peptides. Further application was carried out on different layers of mouse brain micro-sections; our miniPhos method provided quantitative information on protein abundance and phosphosite regulation for the most relevant neurodegenerative diseases, cancers, and signaling pathways in the mouse brain. Surprisingly, the phosphoproteome exhibited more spatial variations than the proteome in the mouse brain. Overall, spatial dynamics of phosphosites are integrated with proteins to gain insights into crosstalk of cellular regulation at different layers, thereby facilitating a more comprehensive understanding of mouse brain development and activity.