Integrated Platform for Large-Scale Quantitative Profiling of Phosphotyrosine Signaling Complexes Based on Cofractionation/Mass Spectrometry and Complex-Centric Algorithm

The
scarcity and dynamic nature of phosphotyrosine (pTyr)-modified
proteins pose a challenge for researching protein complexes with pTyr
modification, which are assembled through multiple protein–protein
interactions. We developed an integrated complex-centric platform
for large-scale quantitative profiling of pTyr signaling complexes
based on cofractionation/mass spectrometry (CoFrac-MS) and a complex-centric
algorithm. We initially constructed a trifunctional probe based on
pTyr superbinder (SH2-S) for specifically binding and isolation of
intact pTyr protein complexes. Then, the CoFrac-MS strategy was employed
for the identification of pTyr protein complexes by integrating ion
exchange chromatography in conjunction with data independent acquisition
mass spectrometry. Furthermore, we developed a novel complex-centric
algorithm for quantifying protein complexes based on the protein complex
elution curve. Utilizing this algorithm, we effectively quantified
216 putative protein complexes. We further screened 21 regulated pTyr
protein complexes related to the epidermal growth factor signal. Our
study engenders a comprehensive framework for the intricate examination
of pTyr protein complexes and presents, for the foremost occasion,
a quantitative landscape delineating the composition of pTyr protein
complexes in HeLa cells.