Integrated and high-throughput approach for sensitive analysis of tyrosine phosphoproteome

ABSTRACT: Tyrosine phosphorylation (pTyr) regulates various signaling pathways under normal and cancerous states. Due to their low abundance, 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 disadvantages of a long sample preparation process, which makes them unsuitable for processing a limited amount of samples, especially in a high-throughput manner. In this study, we developed a 96-well microplate-based approach to integrating all the sample preparation steps starting from cell culture to MS-compatible pTyr peptide enrichment in three consecutive 96-well microplates. By assembling the engineered SH2 domain onto a microplate, non-specific adsorption of phosphopeptides is greatly reduced, which allows us to remove the Ti-IMAC purification and three C18 desalting steps (after digestion, superbinder enrichment, and Ti-IMAC purification), and therefore greatly simplify the entire pTyr peptide enrichment workflow, especially when processing a large number of samples. Starting with 96-well microplate-cultured 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 ug peptides. Importantly, we applied this approach to profile tyrosine kinase inhibitors-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.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): 9606

SUBMITTER: Ruijun Tian  

PROVIDER: MSV000089805 | MassIVE | Sun Jul 03 23:14:00 BST 2022

REPOSITORIES: MassIVE