Abstract
We present the first proteomic analysis on the cellular response to severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection. The differential proteomes of Vero E6 cells with and without infection of the SARS-CoV were resolved and quantitated with two-dimensional differential gel electrophoresis followed by ESI-MS/MS identification. Moreover isotope-coded affinity tag technology coupled with two-dimensional LC-MS/MS were also applied to the differential proteins of infected cells. By combining these two complementary strategies, 355 unique proteins were identified and quantitated with 186 of them differentially expressed (at least 1.5-fold quantitative alteration) between infected and uninfected Vero E6 cells. The implication for cellular responses to virus infection was analyzed in depth according to the proteomic results. Thus, the present work provides large scale protein-related information to investigate the mechanism of SARS-CoV infection and pathogenesis.
Highlights
We present the first proteomic analysis on the cellular response to severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection
As a method based on two-dimensional (2D) electrophoresis, differential gel electrophoresis (DIGE) allows two or three independent samples labeled with different fluorescent dyes such as cyanine-2 (Cy2), cyanine-3 (Cy3), and cyanine-5 (Cy5) to be run in one gel simultaneously and viewed individually using the different fluorescent properties of Cy2, Cy3, and Cy5, circumventing some of the reproducibility problems associated with 2D electrophoresis and providing more accurate quantitative information compared with other staining methods such as silver staining with the dynamic range over 3– 4 orders of magnitude [11, 12]
2D-DIGE Analysis of the SARS-CoV-infected and Uninfected Vero E6 cells—2D-DIGE as a qualitative and quantitative proteomic approach was performed to determine the differential proteomes of the SARS-CoV-infected and uninfected Vero E6 cells
Summary
Materials—Analytical reagent grade chemicals were used throughout unless otherwise stated. DIGE—For DIGE analysis, cellular samples were precipitated overnight with 5 volumes of 50:50:0.1 volumes of ethanol:acetone:acetic acid at Ϫ20 °C and resolubilized in the lysis buffer (7 M urea, 2 M thiourea, 30 mM Tris-Cl, 4% CHAPS). For ICAT analysis, the cellular samples were precipitated and resolubilized in denaturing buffer (6 M guanidine hydrochloride, 100 mM Tris-Cl, pH 8.3). 100 g of the E6 or E6-V protein sample in 80 l of denaturing buffer were reduced at 37 °C for 2 h with 5 mM tributylphosphine (Bio-Rad) and alkylated at 37 °C for 2 h in the dark with ICAT-light and ICAT-heavy reagent, respectively. The ICAT-labeled peptides were purified using the kit of ICATTM Avidin Buffer Pack and Avidin Affinity Cartridge (Applied Biosystems) according to the manufacturer’s guidelines. The protein function and subcellular location annotation was from the Swiss-Prot and TrEMBL protein data base (us.expasy.org/sprot/)
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