Development of a novel nanoflow liquid chromatography-parallel reaction monitoring mass spectrometry-based method for quantification of angiotensin peptides in HUVEC cultures

PeerJ

article

Chuan He¹  Simiao Hu¹  Wanxing Zhou¹ 

[1] The first affiliated hospital of Guangdong Pharmaceutical University, Guangdong, Peopleś Republic of China Guangdong Metabolic Disease Research Center of Integrated Chinese and Western Medicine

关键词: Angiotensin;    High-resolution mass spectrometry;    Human umbilical vein endothelial cells;    Parallel reaction monitoring;    Renin-angiotensin system;   

DOI  :  10.7717/peerj.9941

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

BackgroundThis study aimed to develop an analytical method using liquid chromatography tandem mass spectrometry (LC-MS/MS) for the determination of angiotensin (Ang) I, Ang (1-9), Ang II, Ang (1-7), Ang (1-5), Ang III, Ang IV in human umbilical vein endothelial cell (HUVEC) culture supernatant.MethodsHUVEC culture supernatant was added with gradient concentrations (0.05–1,000 ng/ml) of standard solutions of the Ang peptides. These samples underwent C18 solid-phase extraction and separation using a preconcentration nano-liquid chromatography mass spectrometry system. The target peptides were detected by a Q Exactive quadrupole orbitrap high-resolution mass spectrometer in the parallel reaction monitoring mode. Ang converting enzyme (ACE) in HUVECs was silenced to examine Ang I metabolism.ResultsThe limit of detection was 0.1 pg for Ang II and Ang III, and 0.5 pg for Ang (1-9), Ang (1-7), and Ang (1-5). The linear detection range was 0.1–2,000 pg (0.05–1,000 ng/ml) for Ang II and Ang III, and 0.5–2,000 pg (0.25–1,000 ng/ml) for Ang (1-9) and Ang (1-5). Intra-day and inter-day precisions (relative standard deviation) were <10%. Ang II, Ang III, Ang IV, and Ang (1-5) were positively correlated with ACE expression by HUVECs, while Ang I, Ang (1-7), and Ang (1-9) were negatively correlated.ConclusionThe nanoflow liquid chromatography-parallel reaction monitoring mass spectrometry-based methodology established in this study can evaluate the Ang peptides simultaneously in HUVEC culture supernatant.

【 授权许可】

CC BY