Chemical analysis of C-reactive protein synthesized by human aortic endothelial cells under oxidative stress

Ming Hua Tsai, Chia Liang Chang, Yu San Yu, Ting Yu Lin, Chin Pong Chong, You Sian Lin, Mei Yu Su, Jian Ying Yang, Ting Yu Shu, Xuhai Lu, Chu Huang Chen, Mine Yine Liu

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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.

Original languageEnglish
Pages (from-to)9646-9654
Number of pages9
JournalAnalytical Chemistry
Volume84
Issue number21
DOIs
Publication statusPublished - 2012 Nov 6

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All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

Cite this

Tsai, M. H., Chang, C. L., Yu, Y. S., Lin, T. Y., Chong, C. P., Lin, Y. S., Su, M. Y., Yang, J. Y., Shu, T. Y., Lu, X., Chen, C. H., & Liu, M. Y. (2012). Chemical analysis of C-reactive protein synthesized by human aortic endothelial cells under oxidative stress. Analytical Chemistry, 84(21), 9646-9654. https://doi.org/10.1021/ac302856v