Abstract
The endothelium forms a continuous monolayer at the interface between blood and tissue and contributes significantly to the sensing and transducing of signals between blood and tissue. New blood vessel formation, or angiogenesis, is initiated by the activation of endothelial cells and is an important process required for various pathological and physiological situations. This study used cleavable isotope-coded affinity tag reagents combined with mass spectrometry to investigate the molecular basis of a recently discovered angiogenesis-promoting steroid, sokotrasterol sulfate. Changes in the relative abundances of over 1000 proteins within human endothelial cells treated with sokotrasterol sulfate and vehicle-treated cells were identified and quantitated using this technique. A method that examines the entire ensemble of quantitative measurements was developed to identify proteins that showed a statistically significant change in relative abundance resulting from treatment with sokotrasterol sulfate. A total of 93 proteins was significantly up-regulated, and 37 were down-regulated in response to sokotrasterol sulfate stimulation of endothelial cells. Among the up-regulated proteins, several were identified that are novel to endothelial cells and are likely involved in cell communication and morphogenesis. These findings are consistent with a role for sokotrasterol sulfate in endothelial sprouting.
Highlights
The endothelium forms a continuous monolayer at the interface between blood and tissue and contributes significantly to the sensing and transducing of signals between blood and tissue
Strong Cation Exchange Fractionation of cleavable isotope-coded affinity tag (cICAT)-labeled Peptides— The lyophilized human umbilical vein endothelial cells (HUVECs) cICAT-labeled peptides were dissolved in 170 l of 0.1% formic acid, 25% CH3CN and injected onto a strong cation exchange liquid chromatography (SCXLC) column (1 mm ϫ 150 mm, polysulfoethyl A, PolyLC Inc., Columbia, MD)
Microcapillary Reversed-phase LC-MS/MS of cICAT-labeled Peptides—Ten-centimeter-long RPLC-electrospray ionization (ESI) columns were coupled on line with an ion trap mass spectrometer (LCQ Deca XP, ThermoElectron, San Jose, CA) to analyze the cICATlabeled peptides extracted from the control and sokotrasterol sulfatetreated HUVECs
Summary
Materials—Ammonium bicarbonate (NH4HCO3), ammonium formate (NH4HCO2), guanidine hydrochloride, dibasic sodium phosphate (Na2HPO4), monobasic sodium phosphate (NaH2PO4), sodium chloride (NaCl), ammonium hydroxide (NH4OH), Tris, sodium fluoride (NaF), sodium orthovanadate (Na3VO4), Triton X-100, and phenylmethylsulfonyl fluoride were purchased from Sigma. After washing the column with 10 bed volumes each of 2ϫ PBS, pH 7.2; 1ϫ PBS, pH 7.2; and 50 mM NH4HCO3, pH 8.3, 20% CH3CN, the cICAT-labeled peptides were eluted using 30% CH3CN, 0.4% formic acid and lyophilized to dryness. Strong Cation Exchange Fractionation of cICAT-labeled Peptides— The lyophilized HUVEC cICAT-labeled peptides were dissolved in 170 l of 0.1% formic acid, 25% CH3CN and injected onto a strong cation exchange liquid chromatography (SCXLC) column (1 mm ϫ 150 mm, polysulfoethyl A, PolyLC Inc., Columbia, MD). Microcapillary Reversed-phase LC-MS/MS of cICAT-labeled Peptides—Ten-centimeter-long RPLC-electrospray ionization (ESI) columns were coupled on line with an ion trap mass spectrometer (LCQ Deca XP, ThermoElectron, San Jose, CA) to analyze the cICATlabeled peptides extracted from the control and sokotrasterol sulfatetreated HUVECs. To construct the 10-cm-long RPLC-ESI columns, 75-m-inner diameter fused silica microcapillaries The identified peptides were quantified using XPRESS (ThermoElectron), which calculates the relative abundances (13C9/13C0 in this data set) of peptides based on the area of their extracted ion chromatograms
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