title: Chemical analysis of C-reactive protein synthesized by human aortic endothelial cells under oxidative stress.
abstract: C-reactive protein (CRP) is a clinical biomarker of inflammation, and high levels of CRP correlate with cardiovascular disease. The objectives of this study were to test our hypothesis that oxidized low-density lipoprotein (ox-LDL) induces the release of CRP from human aortic endothelial cells (HAECs) and to optimize several analytical methods to identify CRP released from cultured cells in a model of atherogenic stress. HAECs were incubated with copper-oxidized LDL, and the supernatant was subsequently purified by diethylaminoethyl chromatography and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified an optimal buffer for the elution of CRP, which contained 0.05 M sodium phosphate and 2.0 M NaCl (pH 4.5). Purified CRP was digested with trypsin and subjected to high-performance LC with an optimal mobile phase of acetonitrile-water containing 0.1% formic acid (50:50, v/v) and an optimal mobile phase flow rate of 0.2 mL/min. We identified optimal parameters for MS/MS analysis of CRP, including sheath gas pressure (80 psi), capillary temperature (275 °C), collision energy (25%), tube lens offset (-5 V), auxiliary gas pressure (0 psi), and isolation width of parent ion (m/z value = 3). Characterization of CRP was based on the extracted ion chromatograms and selected multiple-reaction monitoring spectra of three peptides (peptide-1, -2, and -3) derived from trypsin-digested intact CRP standard. CRP peptide-2 and peptide-3 were identified in the supernatant of ox-LDL-treated HAECs. Confirmation of CRP was based on LC-MS/MS and enzyme-linked immunosorbent assay analysis of CRP in purified HAEC supernatant, as well as real-time PCR analysis of CRP mRNA levels in HAECs.
authors.affiliation: Department of Chemistry, National Changhua University of Education, Changhua, Taiwan 50058.
authors.forename: Ming-Hua
authors.initials: MH
authors.lastname: Tsai
authors.forename: Chia-Liang
authors.initials: CL
authors.lastname: Chang
authors.forename: Yu-San
authors.initials: YS
authors.lastname: Yu
authors.forename: Ting-Yu
authors.initials: TY
authors.lastname: Lin
authors.forename: Chin-Pong
authors.initials: CP
authors.lastname: Chong
authors.forename: You-Sian
authors.initials: YS
authors.lastname: Lin
authors.forename: Mei-Yu
authors.initials: MY
authors.lastname: Su
authors.forename: Jian-Ying
authors.initials: JY
authors.lastname: Yang
authors.forename: Ting-Yu
authors.initials: TY
authors.lastname: Shu
authors.forename: Xuhai
authors.initials: X
authors.lastname: Lu
authors.forename: Chu-Huang
authors.initials: CH
authors.lastname: Chen
authors.forename: Mine-Yine
authors.initials: MY
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D001011:Aorta / Q000166:cytology*; D002097:C-Reactive Protein / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002845:Chromatography; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008077:Lipoproteins, LDL / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
chemical_list: D008077:Lipoproteins, LDL; D012333:RNA, Messenger; C099252:oxidized low density lipoprotein; D002097:C-Reactive Protein