High-Throughput and Integrated Chemical Proteomic Approach for Profiling Phosphotyrosine Signaling Complexes

Phosphotyrosine (pTyr)
signaling complexes are important resources
of biomarkers and drug targets which often need to be profiled with
enough throughput. Current profiling approaches are not feasible to
meet this need due to either biased profiling by antibody-based detection
or low throughput by traditional affinity purification-mass spectrometry
approach (AP-MS), as exemplified by our previously developed photo-pTyr-scaffold
approach. To address these limitations, we developed a 96-well microplate-based
sample preparation and fast data independent proteomic analysis workflow.
By assembling the photo-pTyr-scaffold probe into a 96-well microplate,
we achieved steric hindrance-free photoaffinity capture of pTyr signaling
complexes, selective enrichment under denaturing conditions, and efficient
in-well digestion in a fully integrated manner. EGFR signaling complex
proteins could be efficiently captured and identified by using 300
times less cell lysate and 100 times less photo-pTyr-scaffold probe
as compared with our previous approach operated in an Eppendorf tube.
Furthermore, the lifetime of the photo-pTyr-scaffold probe in a 96-well
microplate was significantly extended from 1 week up to 1 month. More
importantly, by combining with high-flow nano LC separation and data
independent acquisition on the Q Exactive HF-X mass spectrometer,
LC–MS time could be significantly reduced to only 35 min per
sample without increasing sample loading amount and compromising identification
and quantification performance. This new high-throughput proteomic
approach allowed us to rapidly and reproducibly profile dynamic pTyr
signaling complexes with EGF stimulation at five time points and EGFR
inhibitor treatment at five different concentrations. We are therefore
optimized for its generic application in biomarkers discovery and
drug screening in a high-throughput fashion.