Integrated and High-Throughput Approach for Sensitive Analysis of Tyrosine Phosphoproteome

Tyrosine phosphorylation (pTyr) regulates various signaling
pathways
under normal and cancerous states. Due to their low abundance and
transient and dynamic natures, systematic profiling of pTyr sites
is challenging. Antibody and engineered binding domain-based approaches
have been well applied to pTyr peptide enrichment. However, traditional
methods have the disadvantage of a long sample preparation process,
which makes them unsuitable for processing limited amount of samples,
especially in a high-throughput manner. In this study we developed
a 96-well microplate-based approach to integrate all the sample preparation
steps starting from cell culture to MS-compatible pTyr peptide enrichment
in three consecutive 96-well microplates. By assembling an engineered
SH2 domain onto a microplate, nonspecific adsorption of phosphopeptides
is greatly reduced, which allows us to remove the Ti-IMAC purification
and three C18 desalting steps (after digestion, pTyr enrichment, and
Ti-IMAC purification) and, therefore, greatly simplifies the entire
pTyr peptide enrichment workflow, especially when processing a large
number of samples. Starting with 96-well microplate-cultured, pervanadate-stimulated
cells, our approach could enrich 21% more pTyr sites than the traditional
serial pTyr enrichment approach and showed good sensitivity and reproducibility
in the range of 200 ng to 200 μg peptides. Importantly, we applied
this approach to profile tyrosine kinase inhibitor-mediated EGFR signaling
pathway and could well differentiate the distinct response of different
pTyr sites. Collectively, the integrated 96-well microplate-based
approach is valuable for profiling pTyr sites from limited biological
samples and in a high-throughput manner.