Proteomic analysis of distinct regions of human carotid atherosclerotic plaques

Protein profiles were determined from different distinct regions of human atherosclerotic plaques, including: internal control, fatty streak, plaque shoulder, plaque centre, and fibrous cap. Protein extracts from all 5 regions, from 3 men and 3 women, were separated by two-dimensional gel electrophoresis (in total 30 2-DE gels). Stained gels were matched by protein spot distribution, and a protein mapping was performed to identify as many protein spots as possible, by MALDI-TOF MS / peptide mass fingerprinting (Voyager DE Pro; Applied Biosystems). This resulted in the identification of 97 protein spots, corresponding to 52 unique protein by accession number. To expand on this nLC-MS/MS (Easy-nLC / LTQ Orbitrap Velos Pro; Thermo Fisher Scientific) experiments were run on additional carotid plaque samples from 10 men and 10 women, using three of the predefined regions; internal control, fatty streak, plaque core. MS/MS analysis resulted in the identification of over 1000 proteins. Human carotid atherosclerotic plaques (n = 26) were obtained from the Linköping Carotid Study, and all methods/protocols described herein have been approved by the local ethics committee (Linköping University Hospital, Linköping, Sweden). Written informed consent was also obtained from all patients.

本研究对人类动脉粥样硬化斑块（atherosclerotic plaques）的不同区域开展蛋白质谱分析，所涉区域包括：内部对照、脂纹（fatty streak）、斑块肩部（plaque shoulder）、斑块核心（plaque centre）及纤维帽（fibrous cap）。研究从3名男性与3名女性的上述5个区域中提取蛋白质，通过双向凝胶电泳（two-dimensional gel electrophoresis, 2-DE）进行分离，共计完成30块双向凝胶电泳胶。对染色后的凝胶依据蛋白质斑点分布进行匹配，并通过基质辅助激光解吸电离飞行时间质谱（matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF MS）结合肽质量指纹谱（peptide mass fingerprinting, PMF）技术，使用Voyager DE Pro质谱仪（应用生物系统公司，Applied Biosystems）开展蛋白质组图谱绘制，以尽可能多地鉴定蛋白质斑点。该流程共鉴定得到97个蛋白质斑点，对应52个具有唯一登录号的蛋白质。为拓展该研究覆盖范围，研究团队针对另外10名男性和10名女性的颈动脉粥样硬化斑块样本，采用预先选定的3个区域（内部对照、脂纹、斑块核心），开展纳流液相色谱-串联质谱（nano-liquid chromatography-tandem mass spectrometry, nLC-MS/MS）实验，实验使用Easy-nLC / LTQ Orbitrap Velos Pro质谱系统（赛默飞世尔科技，Thermo Fisher Scientific）。经串联质谱分析，共鉴定得到超过1000种蛋白质。本研究使用的26例人类颈动脉粥样硬化斑块样本取自林雪平颈动脉研究（Linköping Carotid Study），本研究所述全部方法与实验流程均已通过瑞典林雪平市林雪平大学医院（Linköping University Hospital）当地伦理委员会审批。所有受试者均已签署书面知情同意书。