Signal transductions are critical for cell physiology functions and signaling complexes play major roles in carrying and delivering “signals. These processes occur mainly through protein-protein interaction (PPI) and protein-nucleic acid interaction (PNI). Conventional approaches are usually limited in identifying a specific cellular PPI, or PNI from large amount of a specific protein with limited relative quantification. However, multiple PPIs and PNIs actually occur within a single signaling complex in vivo in a dynamic manner and an effective methodology for accurately detecting these individual complexes for multiple targets are critical for further understanding the in depth mechanisms of cell signaling transduction. Herein we introduce a novel flow-proteometric technique named microchannel for Multi-parameters Analyzing Proteins in Single-complex (mMAPS), which integrates a microfluidics device into single-molecule spectroscopy with three different emission-wavelength detectors to directly detect and analyze each individual signaling complex. Briefly, proteins and/or nucleic acid were labeled by three separate wavelength fluorophores in individual complexes and each complex was examined when it flew through the detection spot in microchannel. Using this approach, we demonstrate various applications by analyzing multiple endogenous PPIs and PNIs in p53, EGFR, HER2, and NFκB complexes from cancer cell lines with the resolution of “single” signaling complex. For instance, using 50,000 cancer cells (HeLa) we have detected and quantified the interaction between three proteins including EGF, EGFR and STAT3. We identified the single protein complexes which contain all three targets (EGF/EGFR/STAT3) after EGF treatment for 30min. EGF/EGFR and EGFR/STAT3 were also been identified as well but we didn't identified EGF/STAT3 interaction. Interestingly, although 20% of detected EGFR were interacted with STAT3 after EGF stimulation, only 3% of EGFR found to interact with EGF in the EGFR complex. In addition, we have also detected the protein complexes containing both proteins and nucleic acid including endogenous p53, TBP1, and genomic DNA fragment and successfully identified p53, TBP1, and genomic DNA in a complex. Quantification results revealed that p53 and TBP1 interaction only occurred in the complexes with genomic DNA. In conclusion, mMAPS provides a powerful platform for direct investigating the composition of individual target protein complexes and reveals the actual quantitative results of specific PTMs, PPIs, and PNIs involved in the signaling complexes, which is important to dissect the detailed dynamics of signal transduction in vivo.

Citation Format: Chao-Kai Chou, Heng-Huan Lee, Pei-Hsiang Tsou, Hirohito Yamaguchi, Ying-Nai Wang, Hong-Jen Lee, Jun Kameoka, Mien-Chie Hung. Flow-proteometric analysis of single signaling complex. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5169. doi:10.1158/1538-7445.AM2013-5169