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

Research output: Contribution to journalArticle

7 Citations (Scopus)

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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Oxidative stress
Endothelial cells
C-Reactive Protein
Chemical analysis
Peptides
formic acid
Trypsin
Gases
Ions
Immunosorbents
Liquid chromatography
Biomarkers
Chromatography
Mass spectrometry
Copper
Lenses
Assays
Buffers

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

Cite this

Tsai, Ming Hua ; Chang, Chia Liang ; Yu, Yu San ; Lin, Ting Yu ; Chong, Chin Pong ; Lin, You Sian ; Su, Mei Yu ; Yang, Jian Ying ; Shu, Ting Yu ; Lu, Xuhai ; Chen, Chu Huang ; Liu, Mine Yine. / Chemical analysis of C-reactive protein synthesized by human aortic endothelial cells under oxidative stress. In: Analytical Chemistry. 2012 ; Vol. 84, No. 21. pp. 9646-9654.
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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.",
author = "Tsai, {Ming Hua} and Chang, {Chia Liang} and Yu, {Yu San} and Lin, {Ting Yu} and Chong, {Chin Pong} and Lin, {You Sian} and Su, {Mei Yu} and Yang, {Jian Ying} and Shu, {Ting Yu} and Xuhai Lu and Chen, {Chu Huang} and Liu, {Mine Yine}",
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Tsai, MH, Chang, CL, Yu, YS, Lin, TY, Chong, CP, Lin, YS, Su, MY, Yang, JY, Shu, TY, Lu, X, Chen, CH & Liu, MY 2012, 'Chemical analysis of C-reactive protein synthesized by human aortic endothelial cells under oxidative stress', Analytical Chemistry, vol. 84, no. 21, pp. 9646-9654. https://doi.org/10.1021/ac302856v

Chemical analysis of C-reactive protein synthesized by human aortic endothelial cells under oxidative stress. / Tsai, Ming Hua; Chang, Chia Liang; Yu, Yu San; Lin, Ting Yu; Chong, Chin Pong; Lin, You Sian; Su, Mei Yu; Yang, Jian Ying; Shu, Ting Yu; Lu, Xuhai; Chen, Chu Huang; Liu, Mine Yine.

In: Analytical Chemistry, Vol. 84, No. 21, 06.11.2012, p. 9646-9654.

Research output: Contribution to journalArticle

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T1 - Chemical analysis of C-reactive protein synthesized by human aortic endothelial cells under oxidative stress

AU - Tsai, Ming Hua

AU - Chang, Chia Liang

AU - Yu, Yu San

AU - Lin, Ting Yu

AU - Chong, Chin Pong

AU - Lin, You Sian

AU - Su, Mei Yu

AU - Yang, Jian Ying

AU - Shu, Ting Yu

AU - Lu, Xuhai

AU - Chen, Chu Huang

AU - Liu, Mine Yine

PY - 2012/11/6

Y1 - 2012/11/6

N2 - 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.

AB - 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.

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