title: Quantitative analysis of a ubiquitin-dependent substrate using capillary electrophoresis with dual laser-induced fluorescence.
abstract: Protein degradation by the ubiquitin-proteasome system (UPS) affects many biological processes. Inhibition of the proteasome has emerged as a potential therapeutic target for cancer treatment. In this study, we developed a method for monitoring the degradation and accumulation of UPS-dependent substrates in cells using CE with dual LIF. We used a green fluorescent protein (GFP)-fusion of the ubiquitin substrate ribophorin 1 (GFP-RPN1) along with red fluorescent protein (RFP) as an internal control to normalize transfection efficiency. Determination of GFP-RPN1 and RFP in cell lysates were performed in an untreated capillary (75 μm × 50 cm) and 100 mM Tris-CHES buffer (pH 9.0) containing 10 mM SDS. GFP-RPN1 and RFP fluorescence were detected at excitation wavelengths of 488 and 635 nm, and emission wavelengths of 520 and 675 nm, respectively, without any interference or crosstalk. The intensity of GFP-RPN1 fluorescence was normalized to that of RFP. Additionally, the proposed approach was used successfully to detect the degradation of GFP-RPN1 and evaluate proteasome inhibitors. These results show that the developed method is effective and promising for rapid and quantitative monitoring of UPS-dependent substrates compared to the current common methods, such as immunoblotting and pulse chase assays.
authors: Molecular Recognition Research Center, Korea Institute of Science and Technology, Seoul, Korea; Department of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul, Korea.
authors: Hyunjung
authors: H
authors: Lee
authors: Eunmi
authors: E
authors: Ban
authors: Eunice EunKyeong
authors: EE
authors: Kim
authors: Young Sook
authors: YS
authors: Yoo
authors: Daekee
authors: D
authors: Lee
authors: Eun Joo
authors: EJ
authors: Song
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects / Q000378:metabolism*; D061988:Proteasome Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D025801:Ubiquitin / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*
chemical_list: D061988:Proteasome Inhibitors; D011506:Proteins; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; D046988:Proteasome Endopeptidase Complex
keywords: CE with dual LIF; GFP-RPN1; RFP; Ubiquitin-dependent substrate; Ubiquitin-proteasome system