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.