ABSTRACT
Oligomerization of proteins and their modified forms (proteoforms) produces functional protein complexes 1,2. Complexoforms are complexes that consist of the same set of proteins with different proteoforms 3. The ability to characterize these assemblies within cells is critical to understanding the molecular mechanisms involved in disease and to designing effective drugs. An outstanding biological question is how proteoforms drive function and oligomerization of complexoforms. However, tools to define endogenous proteoform-proteoform/ligand interactions are scarce 4. Here, we present a native top-down proteomics (nTDP) strategy that combines size-exclusion chromatography, nano liquid-chromatography in direct infusion mode, field asymmetric ion mobility spectrometry, and multistage mass spectrometry to identify protein assemblies (≤70 kDa) in breast cancer cells and in cells that overexpress EGFR, a resistance model of estrogen receptor-α (ER-α) targeted therapies. By identifying ~104 complexoforms from 17 protein complexes, our nTDP approach revealed several molecular features of the breast cancer proteome, including EGFR-induced dissociation of nuclear transport factor 2 (NUTF2) assemblies that modulate ER activity. Our findings show that the K4 and K55 posttranslational modification sites discovered with nTDP differentially impact the effects of NUTF2 on the inhibition of the ER signaling pathway. By characterizing endogenous proteoform-proteoform/ligand interactions, we reveal the molecular diversity of complexoforms, which allows us to propose a model for ER drug discovery in the context of designing effective inhibitors to selectively bind and disrupt the actions of targeted ER complexoforms.
