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Comparison of alternative extraction methods for secretome profiling in human hepatocellular carcinoma cells

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Science China(Life Sciences) 2011年第1期54卷 34-38页

作者： CAO Jing shen ChengPin ZHANG Jun YAO Jun shen huali LIU YinKun LU HaoJie YANG PengYuan Institutes of Biomedical Sciences and Department of Chemistry Fudan University Shanghai 200032 China Liver Cancer Institute Zhongshan Hospital Fudan University Shanghai 200032 China

Secreted proteins are important sources for early detection and diagnosis of disease, and as such have received considerable attention. The extraction of low concentration proteins from large volumes of culture media, which are rich in salts and other compounds that interfere with most proteomics techniques, presents a problem for secretome studies. Ultrafiltration, precipitation, and dialysis are three major extraction methods that can be used to overcome this problem. The present study for the first time, compared the merits and shortcomings of these three methods, without bias. Centrifugal ultrafiltration provided the best extraction efficiency, and precipitation provided the highest number of identifiable proteins. The three methods yielded closely related, but different, information on the secretome; thus, they should be considered complementary or, at least, supplementary methods. Three hundred and sixty unique proteins were identified, including 211 potential secreted proteins. Compared with previous studies, this study also identified 42 new secreted proteins. The present study not only offers a reference for the selection of secretome extraction methods, but also expands the secretome database for the investigation of hepatocellular carcinoma.

关键词： hepatocellular carcinoma secretome protein extraction ultrafiltration precipitation dialysis LC-MS/MS

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Science China Chemistry 2016年第3期59卷 380-382页

作者： Li Feng Hong Li Yang Zhang huali shen Hongxiu Yu Fan Zhong Lunxiu Qin Fuchu He Pengyuan Yang Zhaoyou Tang Minhang Hospital Fudan UniversityShanghai201199China Department of Chemistry Fudan UniversityShanghai200433China Institutes of Biomedical Sciences Fudan UniversityShanghai200032China Zhongshan Hospital Fudan UniversityShanghai200032China

Recently, we have analyzed expressional transcriptome and proteome for a number of cancer cell lines, including 8 hepatocellular carcinoma （HCC） ones [1,2]. These 8 HCC cell lines consist of 6 metastatic （all with mutant p53） and 2 non-metastatic （with a mutant and a lost p53） ones, being all HBV traceable for relatively close genetic-backgrounds. We have analyzed the related dataset and obtained transcrip- tome （16353 genes） and proteome （7861 proteins） of these 8 HCC cell lines, and explored a group of up-regulated and down-regulated genes [3].

关键词： Cell culture Chromosomes Genes Pathology

Multiomics Integration Reveals the Landscape of Differential Response to High Fat Stimulation in the Pathogenesis of Atherosclerosis

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Multiomics Integration Reveals the Landscape of Differential...

Functional lipidomics: palmitic acid impairs hepatocellular carcinoma development by modulating membrane fluidity and glucose metabolism

中国化学会第十三届全国分析化学年会

作者： Xie Juanjuan shen huali Yang Pengyuan Institute of Biomedical Science Fudan University Department of Chemistry Fudan University

＜正＞Atherosclerosis （AS） is the main cause of coronary heart disease and other cardiovascular diseases that severely threatening human health worldwide1). AS is a chronic and progressive multifocal arterial disease associated with lipid deposition, inflammatory response and abnormal remodeling of the arterial wall2). The complicated disease process of AS hampered research works from fully uncover the pathological mechanisms. In this study, we adopted quantitative proteomic and lipidomics analysis on the aortic vessels of the APOE-/- and WT mice with normal diet （ND） or high fat diet （HFD）. More than 6, 200 proteins and 1000 lipids have been quantified in total.

关键词： Atherosclerosis proteomics Lipidomics ApoE-/-mouse model

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Functional lipidomics: palmitic acid impairs hepatocellular ...

具核梭杆菌具核亚种蛋白质组及其在癌症pH环境下的差异蛋白质组分析

中国化学会第十三届全国分析化学年会

作者： Ling Lin huali shen Pengyuan Yang Institutes of Biomedical Sciences of Shanghai Medical School Fudan University Departments of Chemistry Fudan University

Lipids are essential cellular components and energy sources of living organisms, altered lipid composition is increasingly recognized as a signature of cancer. We performed the lipidomic analysis in a series of hepatocellular carcinoma（HCC） cells and identified over 1700 intact lipids originating from 3 major lipid categories. Comparative lipidomic screening revealed 93 significantly changed lipids and decreased palmitic acyl（C16:0）-containing glycerophospholipids（GPs） were positively associated with metastatic abilities of HCC cells. Furthermore, both in vitro and in vivo experiments designated that C16:0 incubation specifically reduced malignant cell proliferation, impaired cell invasiveness, and suppressed tumor growth in mouse xenograft models. Biochemical experiments demonstrated that C16:0 treatment decreased cell membrane fluidity and limited glucose metabolism. Phosphoproteomics approach further revealed such C16:0 incubation attenuated phosphorylation levels of m TOR and Stat3 pathway proteins. These data collectively provide the essential theoretical and experimental basis for diagnosing and treating HCCs by targeting the altered lipid metabolism.

关键词： lipidomics hepatocellular carcinoma metastasis palmitic acid glucose homeostasis

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化学学报 2015年第4期73卷 337-342页

作者：殷薛飞刘晓慧申华莉金红杨芃原复旦大学化学系上海200433 复旦大学生物医学研究院上海200032

利用LC-MS/MS对***的蛋白质表达谱进行了深入研究,共鉴定到1198个蛋白质的表达,占基因组58%的编码基因.通过在p H 6.5(癌症组织p H环境)和p H 7.5(正常组织p H环境)两种条件下***蛋白质表达的定量蛋白质组学分析,发现处于癌症微环境p H... 详细信息

利用LC-MS/MS对***的蛋白质表达谱进行了深入研究,共鉴定到1198个蛋白质的表达,占基因组58%的编码基因.通过在p H 6.5(癌症组织p H环境)和p H 7.5(正常组织p H环境)两种条件下***蛋白质表达的定量蛋白质组学分析,发现处于癌症微环境p H条件下蛋白质表达的变化明显集中于金属离子转运,硫胺素代谢,糖代谢等过程和功能.产丁酸发酵的能量代谢通路中的七个代谢酶在蛋白质组数据中找到,其中有五个在癌症p H条件下显著下调,这五个酶包含了丁酸盐代谢的整个过程,这为结直肠癌患者肠道丁酸盐含量降低提供了新的证明.此外,癌症p H条件下***的丁酸代谢受到抑制,造成产丁酸能力下降,可能是其促进结直肠癌发展在代谢水平上的原因之一.

关键词：具核梭杆菌蛋白质组非标定量 pH环境丁酸代谢

Proteomic Analysis for Metastasis-associated of Human Hepatocellular Carcinoma

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Proteomic Analysis for Metastasis-associated of Human Hepato...

高效液相色谱-质谱法对冠心病患者血清的脂质组学研究及其临床意义

第四届国际华夏学者分析化学研讨会

作者： Pengyuan Yang huali shen Yinkun Liu Zhaoyou Tang Institutes for Biomedical Sciences Department of Chemistry Research Institute for Liver Cancer Fudan University Shanghai 200032 China

＜正＞The prognosis of Hepatocellular carcinoma （HCC） remains dismal mainly because of its late diagnosis, especially in patients with coexisting chronic liver diseases. In most cases human liver cancer are HCC with highly metastatic ahility such that the early prediction or diagnosis and effective treatment are the key point in research. A human HCC nude mice model （LCI-D20） with high metastasis potential have successfully established previously by us. It is worth to analyze the relativity of all expressed protein profile in the LCI-D20 HCC nude mice model in order to understand better the tumor biology of HCC. We have identified an expressed proteome for human hepatocellular carcinoma （HCC） in nude mice model. With advanced proteomic technologies, a proteomic. analysis has been carried out, and the identified proteins were classified bioinformatically respective to their function, biological process and intracellular localization. Some important proteins founded in HCC, e.g., metabolism enzymes, proteins regulating cell motility, signaling proteins, and heat shock proteins, are discussed in terms of their metastasis.

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理化检验（化学分册） 2021年第1期57卷 32-35页

作者：王真昕申华莉杨芃原复旦大学上海医学院生物医学研究院上海200032 复旦大学附属中山医院检验科上海200032 复旦大学附属闵行医院上海201199 复旦大学化学系上海200433

采用高效液相色谱-质谱法(HPLC-MS)对冠状动脉慢性完全堵塞(CTO)患者在不同患病阶段的血清试样进行了脂质组学研究。试验采集了CTO疑似人群、CTO患者及CTO患者经皮冠状动脉介入治疗(PCI)手术后24h和72h的血清,分别采用氯仿和体积比为25... 详细信息

采用高效液相色谱-质谱法(HPLC-MS)对冠状动脉慢性完全堵塞(CTO)患者在不同患病阶段的血清试样进行了脂质组学研究。试验采集了CTO疑似人群、CTO患者及CTO患者经皮冠状动脉介入治疗(PCI)手术后24h和72h的血清,分别采用氯仿和体积比为25∶75的乙酸乙酯-异辛烷混合溶液提取脂质,离心分离后,上清液用于Shotgun法(MS)和多反应监测(MRM)法(HPLC-MS)分析。样本直接进样电喷雾(ESI)质谱分析,用于Shotgun法进行广谱的脂质组学分析鉴定。通过Analyst 1.6软件提取质谱数据,并通过生物信息学工具"Lipid MS Predict"确认脂质分子,总共得到1504种脂质分子,并利用MRM法确认其中有临床意义的631个脂质分子。通过生物信息学分析,得到对于CTO具有诊断价值的包括胆固醇酯CE 16∶1和溶血磷脂酰乙醇胺LPE 18∶1在内的9个标志性脂质分子。结合临床回访结果和脂质组学研究数据证实,CTO患者手术治疗后的预后情况和磷脂酸、磷脂酰胆碱、胆固醇酯等3类脂质的含量变化有关。确定的脂质标志物应用于临床诊断早期冠心病和评估手术治疗后的风险,具有显著而明确的临床医学价值。

关键词：高效液相色谱-质谱法脂质组学冠状动脉慢性完全堵塞临床诊断

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糖蛋白结构质谱解析的样品前处理

色谱 2007年第5期25卷 623-627页

作者：周玮刘晓慧周新文申华莉杨芃原复旦大学生物医学研究院上海200032

基于高分辨率、高精度、高灵敏度及良好表现力的多级质谱,以标准糖蛋白辣根过氧化物酶(HRP)、核糖核酸酶B(RNaseB)为对象,探索了糖蛋白质谱分析前样品处理的技术流程。通过对糖蛋白样品前处理的各个步骤(包括蛋白变性方法(RNaseB适合化... 详细信息

基于高分辨率、高精度、高灵敏度及良好表现力的多级质谱,以标准糖蛋白辣根过氧化物酶(HRP)、核糖核酸酶B(RNaseB)为对象,探索了糖蛋白质谱分析前样品处理的技术流程。通过对糖蛋白样品前处理的各个步骤(包括蛋白变性方法(RNaseB适合化学变性,HRP适合热变性)、酶的选择及条件(RNaseB适用内蛋白酶Lys-C酶解,HRP适用胰蛋白酶酶解,酶解时间控制在12～16h)、提肽液的组成(对于低丰度糖蛋白用50%乙腈-5%三氟乙酸提取)、基质的组成、点样方法("三明治"式))进行了优化,形成了简单易行、无需糖肽富集标记的糖蛋白溶液酶解-质谱分析和胶上分离酶解-质谱分析的技术路线,为实际样品体系中糖蛋白的结构解析提供了可行的方法。

关键词：糖蛋白结构质谱解析样品前处理条件优化

数字化情境下制造企业跨界行为、资源获取与持续竞争优势

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上海立信会计金融学院学报 2022年第1期34卷 95-109页

作者：薛芳赵晓康沈华礼东华大学旭日工商管理学院上海200051 上海立信会计金融学院外国语学院上海201620 萍乡学院商学院江西萍乡337055

数字技术大大降低了资源流通成本,颠覆了人们对组织边界的认知,有力地推动了企业跨组织资源的获取及整合。文章构建跨界行为、资源获取与持续竞争优势理论模型,对127家制造企业问卷调查数据进行分析,研究发现:跨界行为的广度与深度对企... 详细信息

数字技术大大降低了资源流通成本,颠覆了人们对组织边界的认知,有力地推动了企业跨组织资源的获取及整合。文章构建跨界行为、资源获取与持续竞争优势理论模型,对127家制造企业问卷调查数据进行分析,研究发现:跨界行为的广度与深度对企业持续竞争优势具有显著正向影响;跨界广度和跨界深度对企业信息知识获取具有显著正向影响,跨界深度对企业声誉获取具有正向影响,但是跨界广度对企业声誉获取的影响并不显著;信息知识获取与声誉获取对企业持续竞争优势获取具有正向影响;信息知识获取在跨界广度、跨界深度与企业持续竞争优势的关系中具有显著中介作用。文章旨在推动数字化情境下跨界行为的相关研究,为企业通过跨界行为推动资源跨界整合、扩大企业虚拟边界、实现数字化转型提供理论支持。

关键词：数字化跨界行为资源获取持续竞争优势