Deep Phosphotyrosine Proteomics by Optimization of Phosphotyrosine Enrichment and MS/MS Parameters

Phosphorylation is a major post-translational
modification that
regulates protein function, with phosphotyrosine (pY) modifications
being implicated in oncogenesis. However, global profiling of pY statuses
without treatment with a tyrosine phosphatase inhibitor such as pervanadate
is still challenging due to the low occupancy of pY sites. In this
study, we greatly improved the identification of pY sites by liquid
chromatography–tandem mass spectrometry (LC–MS/MS) by
optimization of both the pY-immunoprecipitation (pY-IP) protocol and
the LC–MS/MS parameters. Our highly sensitive method reproducibly
identified more than 1000 pY sites from 8 mg of protein lysate without
the need for tyrosine phosphatase inhibitor treatment. Furthermore,
>30% of the identified pY sites were not assigned in the PhosphositePlus
database. We further applied our method to the comparison of pY status
between PC3 cells with and without treatment using the epidermal growth
factor receptor (EGFR) inhibitor Erlotinib. Under Erlotinib treatment,
we observed not only a decrease in well-known modes of EGFR downstream
signaling but also modulations of kinases that are not relevant to
the EGFR cascade, such as PTK6 and MAPK13. Our newly developed method
for pY proteomics has the potential to reveal unknown pY signaling
modes and to identify novel kinase anticancer targets.