Objective To research a protein chip method which can simultaneously quantitative detect β-Lactoglobulin （β-L） and Lactoferrin （Lf） at one time. Methods Protein chip printer was used to print both anti-β-L anti...Objective To research a protein chip method which can simultaneously quantitative detect β-Lactoglobulin （β-L） and Lactoferrin （Lf） at one time. Methods Protein chip printer was used to print both anti-β-L antibodies and anti-Lf antibodies on each block of protein chip. And then an improved sandwich detection method was applied while the other two detecting antibodies for the two antigens were added in the block after they were mixed. The detection conditions of the quantitative detection for simultaneous measurement of β-L and Lf with protein chip were optimized and evaluated. Based on these detected conditions, two standard curves of the two proteins were simultaneously established on one protein chip. Finally, the new detection method was evaluated by using the analysis of precision and accuracy. Results By comparison experiment, mouse monoclonal antibodies of the two antigens were chosen as the printing probe. The concentrations of β-L and Lf probes were 0.5 mg/mL and 0.5 mg/mL, respectively, while the titers of detection antibodies both of β-L and Lf were 1：2,000. Intra- and inter-assay variability was between 4.88% and 38.33% for all tests. The regression coefficients of protein chip comparing with ELISA for β-L and Lf were better than 0.734, and both of the two regression coefficients were statistically significant （r = 0.734, t = 2.644, P = 0.038; and r = 0.774, t = 2.998, P = 0.024）. Conclusion A protein chip method of simultaneously quantitative detection for β-L and Lf has been established and this method is worthy in further application.展开更多
BACKGROUND： Studies have reported the combined use of two-dimensional gel electrophoresis and mass spectrometry to detect differentially expressed proteins in the rat brainstem following brain injury. However, the de...BACKGROUND： Studies have reported the combined use of two-dimensional gel electrophoresis and mass spectrometry to detect differentially expressed proteins in the rat brainstem following brain injury. However, the detected differential proteins often exhibit low sensitivity and high relative molecular weight. Although protein chip technology is thought to compensate for these inadequacies, no related studies or results have been reported. OBJECTIVE： To propose the application of weak cation exchange protein chips in combination with mass spectrometry for determining protein expression profiles and characteristics in the brainstem following closed brain injury. DESIGN, TIME AND SETTING： Randomized, controlled, animal experiments utilizing proteomics were performed from June 2007 to December 2008 in the Proteomics Laboratory, Medical College of Chinese People＇s Armed Police Force. MATERIALS： Weak cation exchange 2 protein chip, Ciphergen Proteinchip System （PBS-IIC）. METHODS： A total of 72 rats were randomly assigned to two groups： sham-surgery （n = 12） and injury （n = 60）. A closed traumatic brain injury model caused by falling object was replicated in the injury group, which was then subdivided into five subgroups according to different time points after injury： 4, 8, 12, 24, and 48 hours, with 12 rats in each subgroup. In the sham-surgery group, only the skin was removed and the stainless steel pad was fixed to the skull. MAIN OUTCOME MEASURES： The brain injury rats were sacrificed at 4, 8, 12, 24, and 48 hours after injury, respectively, and the control rats were sacrificed at 24 hours. Pathological changes in the brainstem were determined using hematoxylin-eosin staining, and differential protein expression in the brainstem was detected using a weak cation exchange 2 protein chip and protein chip reader. RESULTS： In the sham-surgery group, cells appeared normal. However, in the brain injury group, some brainstem neurons exhibited pyknosis, with reduced numbers of Nissl bodies in the cytoplasm swollen cell bodies and nuclei, irregular staining in the cytoplasm, and decreased numbers of neurons. Results from weak cation exchange 2 protein chip detection demonstrated that, compared with the sham-surgery group, the expression profiles of 2 proteins were altered in the brainstem of the injury group. At 12, 24, and 48 hours after injury, expression was increased （P 〈 0.01 ）. The mass charge ratio （M/Z） of 7 862 differentially expressed proteins was greater in the sham-surgery group compared with 12 and 24 hours after injury （P 〈 0.05）. CONCLUSION： The combined method of weak cation exchange 2 protein chip and mass spectrometry detected differential protein expression in the brainstem following closed brain injury in the rats, which suggested that closed brain injury induced altered protein expression profiles in the brainstem.展开更多
The proteomics of the differential protein expressions in human glioma cell line U251 cells infected with human cytomegalovirus （HCMV） was investigated at the protein level by using the surface enhanced laser desorp...The proteomics of the differential protein expressions in human glioma cell line U251 cells infected with human cytomegalovirus （HCMV） was investigated at the protein level by using the surface enhanced laser desorption/ionization （SELDI） protein chip system in order to develop a method of study for the pathogenesis of HCMV infection. In this study, the cultured U251 cells were infected with HCMV in good condition and the supernatants of lysates and the extracellular fluids of the cultivated infected cells were quantitatively defined for the expressed proteins. The proteomics of the differential protein expression in cells before and after infection was analyzed by WCX2 arrays on the protein chip reader. It was demonstrated that the eytopathic effects of infected cells appeared on the 5th day after infection, however, the differential protein expression was evident at 6 h after infection as revealed by RT-PCR and mass spectrometry. The protein peaks captured from different batches of samples, from the same sample detected with different arrays or for the different times were all equivalent. With the molecular weight range from 2000 Da to 3000 Da, chip captured 82 peaks from the intracellular fluids and 11 protein peak from the cellular fluid in which compared with the control group, the protein peaks with molecular weight of 13 536.3 Da, 10 046.1 Da and 17 106.2 Da were close to those of β-amyloid protein, caspase-1 precursor and LPS-induced TNF-α factor respectively, which showed brief up-regulation 4 h after infection, and continued to raise 48 h later. These results infer that these proteins may be related to the apoptosis induced by HCMV infection, thus suggesting that the apoptosis induced by HCMV infection may play a role in the pathogenesis of HCMV infection.展开更多
The purpose of this study was to develop a protein chip technique based on receptor binding assays to measure bioavailable serum sex hormone levels (BSSHL). 224 aging healthy Chinese were investigated to get the refer...The purpose of this study was to develop a protein chip technique based on receptor binding assays to measure bioavailable serum sex hormone levels (BSSHL). 224 aging healthy Chinese were investigated to get the referenced values of BSSHL for the first time. In the assays recombined sex hormone receptor proteins were jointed to polysaccharide coated slides to make protein chip,; the dose-dependence curves of sex hormone on chip were prepared. The data showed that this method had good precision (CV>16%); accuracy (Bias>10%),; the sensitivity could reach 1 pmol/L. From the results, BSSHL of men; women declined with aging, but no significant differences were observed. The BSSHL of aging men were higher than those of women. The bioavailable serum;rogen level of men was 52–112 pmol/L, women’s was 3–70 pmol/L; the whole group was 41.9–81.4 pmol/L. The bioavailable serum estrogen level of men was 0.8–3.0 pmol/L, women’s was 1.2–2.5 pmol/L; the whole group was 0.6–2.64 pmol/L. Based on the assays, BSSHL measurement by protein chip can meet the needs of epidemiological studies in terms of speed, accuracy; sample volume required,; was helpful in quantitative assessment of aging people’s health.展开更多
Objective： To study the expressive characteristics of SKP2 protein in lung carcinoma and its implication for prognosis. Methods： The expression of SKP2 protein was detected in 89 non small cell lung carcinoma, 13 sm...Objective： To study the expressive characteristics of SKP2 protein in lung carcinoma and its implication for prognosis. Methods： The expression of SKP2 protein was detected in 89 non small cell lung carcinoma, 13 small cell lung carcinoma, 10 lung benign lesion tissues by Tissue Chip and Immunohistochemistry technology. Results： The positive rate of SKP2 protein staining was （23.52±13.57）% in non small cell lung carcinoma and （53.85±12.26）% in small cell lung carcinoma, which were significantly higher than （2.91±1.27）% in lung benign lesion tissues. It was highest in small cell lung carcinoma and lowest in lung benign lesion tissues, with a significant difference between them （P=0.000）. The expressive level of SKP2 protein in lung carcinoma tissues was closely related to cell differentiation, lymph node metastasis and pathological types, but not to age, sex, smoking history, tumor site and size, and TNM staging. The survival analysis revealed that the 5-year survival rate of lung carcinoma patients was lower in SKP2 protein positive expression group than that in negative expression group （P1=0.003/0.002; r=-0.275, P2=0.005）. Conclusion. The positive expression of SKP2 protein is higher in lung carcinoma than in lung benign lesion tissues, in particular, much higher in small cell lung carcinoma. In lung carcinoma, its expressive level was closely related to cell differentiation, lymph node metastasis and pathological types. Moreover, it may be an independent factor to prognosis of patients with lung carcinoma.展开更多
This study describes the development of a universal phosphorylated peptide-binding protein designed to simultaneously detect serine, threonine and tyrosine kinases. The Escherichia
染色体免疫共沉淀测序(Chromatin immunoprecipitation followed by sequencing,ChIP-seq)是研究DNA-蛋白质互作的有力工具,被广泛用于RNA聚合酶、转录因子和组蛋白修饰等在基因组上的精确定位。近年来,在ChIP-seq技术的基础上,科学家...染色体免疫共沉淀测序(Chromatin immunoprecipitation followed by sequencing,ChIP-seq)是研究DNA-蛋白质互作的有力工具,被广泛用于RNA聚合酶、转录因子和组蛋白修饰等在基因组上的精确定位。近年来,在ChIP-seq技术的基础上,科学家提出了一系列研究DNA-蛋白质互作的技术方法,提高了测序分辨率,降低了实验成本,极大推动了表观基因组学的发展。本文综述了多种DNA-蛋白质互作研究技术的原理及其应用场景,介绍了在单细胞水平上研究DNA-蛋白质互作的实现方法,并展望其未来发展的方向。展开更多
Objective： To study the expressive characteristics of SKP2 protein in non-small cell lung carcinoma and it is affection to NSCLC patients＇ prognosis. Methods： The expression of SKP2 protein was detected in 89 NSCLC...Objective： To study the expressive characteristics of SKP2 protein in non-small cell lung carcinoma and it is affection to NSCLC patients＇ prognosis. Methods： The expression of SKP2 protein was detected in 89 NSCLC, 5 benign lung neoplasmas, 5 normal bronchus and lung tissues by Tissue Chip and immunohistochemistry technology. Results： The positive rate of SKP2 protein staining was （23.52±13.57）% in NSCLC tissues, significantly higher than that in benign lung neoplasmas, normal brochus and lung tissues （2.91±1.27）% （P=0.0000〈0.001）. The expressive level of SKP2 protein in NSCLC tissues was closely related to cell differentiation （PI=0.000〈0.001）, but not to age, sex, smoking history, pathological type, site, size, lymph node metastasis and TNM stage （each Pl〉0.05）. The survival analysis displayed that the NSCLC patients＇ 5 years survival rate was lower in positive expression group than that in negative expression group （P1=0.042/0.031〈0.05; r=-0.186, P2=0.000〈0.001）. Conclusion： The positive expression of SKP2 protein may play an enhancement role in the occurrence and development of NSCLC. Moreover, it may be a bad indicator to NSCLC patients＇ prognosis.展开更多
目的基于基因芯片数据分析PHD锌指蛋白19(PHD finger protein 19,PHF19)在非小细胞肺癌(non-small cell lung cancer,NSCLC)中的表达及预后价值。方法下载癌症基因组图谱(The Cancer Genome Atlas,TCGA)肺癌患者数据并分析PHF19在肺癌...目的基于基因芯片数据分析PHD锌指蛋白19(PHD finger protein 19,PHF19)在非小细胞肺癌(non-small cell lung cancer,NSCLC)中的表达及预后价值。方法下载癌症基因组图谱(The Cancer Genome Atlas,TCGA)肺癌患者数据并分析PHF19在肺癌中表达,通过基因表达数据库(Gene Expression Omnibus,GEO)下载数据集GSE30219,GSE50081筛选患者分别纳入训练集和验证集,并分析PHF19表达与肺癌患者性别、年龄、肿瘤临床分期、病理类型与无病生存率(disease free survival,DFS)、总生存率(overall survival,OS)的关系,通过在线数据库对肺癌患者PHF19及共表达相关基因进行基因本体论与京都基因和基因组百科全书(Gene Ontology and Kyoto Encyclopedia of Genes and Genomes,GO-KEGG)富集分析、免疫浸润分析。结果TCGA、GEO数据显示PHF19在肺癌中高表达(P<0.001),PHF19表达与肿瘤分期相关。PHF19低表达组的NSCLC患者较高表达组具有更长的DFS及OS(P均<0.05)。多因素COX回归分析显示PHF19为NSCLC患者独立预后影响因素(P<0.05)。绘制列线图预测肺癌患者的生存率并验证C指数表明模型准确度良好。基因富集分析显示PHF19高表达主要与细胞周期、细胞核、染色质等相关。免疫浸润分析显示PHF19与免疫细胞浸润密切相关。结论PHF19可作为预测NSCLC预后的指标,PHF19高表达是NSCLC不良预后的独立预测因子,可能是其治疗的新的靶点。展开更多
基金Sponsored by the Young Scholar Scientific Research Foundation of China CDC[2015A202]:The establishment of testing platform of quantitatively detecting main protein of cow milk by using protein chip technique
文摘Objective To research a protein chip method which can simultaneously quantitative detect β-Lactoglobulin （β-L） and Lactoferrin （Lf） at one time. Methods Protein chip printer was used to print both anti-β-L antibodies and anti-Lf antibodies on each block of protein chip. And then an improved sandwich detection method was applied while the other two detecting antibodies for the two antigens were added in the block after they were mixed. The detection conditions of the quantitative detection for simultaneous measurement of β-L and Lf with protein chip were optimized and evaluated. Based on these detected conditions, two standard curves of the two proteins were simultaneously established on one protein chip. Finally, the new detection method was evaluated by using the analysis of precision and accuracy. Results By comparison experiment, mouse monoclonal antibodies of the two antigens were chosen as the printing probe. The concentrations of β-L and Lf probes were 0.5 mg/mL and 0.5 mg/mL, respectively, while the titers of detection antibodies both of β-L and Lf were 1：2,000. Intra- and inter-assay variability was between 4.88% and 38.33% for all tests. The regression coefficients of protein chip comparing with ELISA for β-L and Lf were better than 0.734, and both of the two regression coefficients were statistically significant （r = 0.734, t = 2.644, P = 0.038; and r = 0.774, t = 2.998, P = 0.024）. Conclusion A protein chip method of simultaneously quantitative detection for β-L and Lf has been established and this method is worthy in further application.
基金the National Natural Science Foundation of China, No.30471934
文摘BACKGROUND： Studies have reported the combined use of two-dimensional gel electrophoresis and mass spectrometry to detect differentially expressed proteins in the rat brainstem following brain injury. However, the detected differential proteins often exhibit low sensitivity and high relative molecular weight. Although protein chip technology is thought to compensate for these inadequacies, no related studies or results have been reported. OBJECTIVE： To propose the application of weak cation exchange protein chips in combination with mass spectrometry for determining protein expression profiles and characteristics in the brainstem following closed brain injury. DESIGN, TIME AND SETTING： Randomized, controlled, animal experiments utilizing proteomics were performed from June 2007 to December 2008 in the Proteomics Laboratory, Medical College of Chinese People＇s Armed Police Force. MATERIALS： Weak cation exchange 2 protein chip, Ciphergen Proteinchip System （PBS-IIC）. METHODS： A total of 72 rats were randomly assigned to two groups： sham-surgery （n = 12） and injury （n = 60）. A closed traumatic brain injury model caused by falling object was replicated in the injury group, which was then subdivided into five subgroups according to different time points after injury： 4, 8, 12, 24, and 48 hours, with 12 rats in each subgroup. In the sham-surgery group, only the skin was removed and the stainless steel pad was fixed to the skull. MAIN OUTCOME MEASURES： The brain injury rats were sacrificed at 4, 8, 12, 24, and 48 hours after injury, respectively, and the control rats were sacrificed at 24 hours. Pathological changes in the brainstem were determined using hematoxylin-eosin staining, and differential protein expression in the brainstem was detected using a weak cation exchange 2 protein chip and protein chip reader. RESULTS： In the sham-surgery group, cells appeared normal. However, in the brain injury group, some brainstem neurons exhibited pyknosis, with reduced numbers of Nissl bodies in the cytoplasm swollen cell bodies and nuclei, irregular staining in the cytoplasm, and decreased numbers of neurons. Results from weak cation exchange 2 protein chip detection demonstrated that, compared with the sham-surgery group, the expression profiles of 2 proteins were altered in the brainstem of the injury group. At 12, 24, and 48 hours after injury, expression was increased （P 〈 0.01 ）. The mass charge ratio （M/Z） of 7 862 differentially expressed proteins was greater in the sham-surgery group compared with 12 and 24 hours after injury （P 〈 0.05）. CONCLUSION： The combined method of weak cation exchange 2 protein chip and mass spectrometry detected differential protein expression in the brainstem following closed brain injury in the rats, which suggested that closed brain injury induced altered protein expression profiles in the brainstem.
基金This work was supported by National Natural Science Foundation of China(No.30471527 and No.30540075)partly supported by Mr.Tai Scholar Construction Engineering Foundation.
文摘The proteomics of the differential protein expressions in human glioma cell line U251 cells infected with human cytomegalovirus （HCMV） was investigated at the protein level by using the surface enhanced laser desorption/ionization （SELDI） protein chip system in order to develop a method of study for the pathogenesis of HCMV infection. In this study, the cultured U251 cells were infected with HCMV in good condition and the supernatants of lysates and the extracellular fluids of the cultivated infected cells were quantitatively defined for the expressed proteins. The proteomics of the differential protein expression in cells before and after infection was analyzed by WCX2 arrays on the protein chip reader. It was demonstrated that the eytopathic effects of infected cells appeared on the 5th day after infection, however, the differential protein expression was evident at 6 h after infection as revealed by RT-PCR and mass spectrometry. The protein peaks captured from different batches of samples, from the same sample detected with different arrays or for the different times were all equivalent. With the molecular weight range from 2000 Da to 3000 Da, chip captured 82 peaks from the intracellular fluids and 11 protein peak from the cellular fluid in which compared with the control group, the protein peaks with molecular weight of 13 536.3 Da, 10 046.1 Da and 17 106.2 Da were close to those of β-amyloid protein, caspase-1 precursor and LPS-induced TNF-α factor respectively, which showed brief up-regulation 4 h after infection, and continued to raise 48 h later. These results infer that these proteins may be related to the apoptosis induced by HCMV infection, thus suggesting that the apoptosis induced by HCMV infection may play a role in the pathogenesis of HCMV infection.
文摘The purpose of this study was to develop a protein chip technique based on receptor binding assays to measure bioavailable serum sex hormone levels (BSSHL). 224 aging healthy Chinese were investigated to get the referenced values of BSSHL for the first time. In the assays recombined sex hormone receptor proteins were jointed to polysaccharide coated slides to make protein chip,; the dose-dependence curves of sex hormone on chip were prepared. The data showed that this method had good precision (CV>16%); accuracy (Bias>10%),; the sensitivity could reach 1 pmol/L. From the results, BSSHL of men; women declined with aging, but no significant differences were observed. The BSSHL of aging men were higher than those of women. The bioavailable serum;rogen level of men was 52–112 pmol/L, women’s was 3–70 pmol/L; the whole group was 41.9–81.4 pmol/L. The bioavailable serum estrogen level of men was 0.8–3.0 pmol/L, women’s was 1.2–2.5 pmol/L; the whole group was 0.6–2.64 pmol/L. Based on the assays, BSSHL measurement by protein chip can meet the needs of epidemiological studies in terms of speed, accuracy; sample volume required,; was helpful in quantitative assessment of aging people’s health.
文摘Objective： To study the expressive characteristics of SKP2 protein in lung carcinoma and its implication for prognosis. Methods： The expression of SKP2 protein was detected in 89 non small cell lung carcinoma, 13 small cell lung carcinoma, 10 lung benign lesion tissues by Tissue Chip and Immunohistochemistry technology. Results： The positive rate of SKP2 protein staining was （23.52±13.57）% in non small cell lung carcinoma and （53.85±12.26）% in small cell lung carcinoma, which were significantly higher than （2.91±1.27）% in lung benign lesion tissues. It was highest in small cell lung carcinoma and lowest in lung benign lesion tissues, with a significant difference between them （P=0.000）. The expressive level of SKP2 protein in lung carcinoma tissues was closely related to cell differentiation, lymph node metastasis and pathological types, but not to age, sex, smoking history, tumor site and size, and TNM staging. The survival analysis revealed that the 5-year survival rate of lung carcinoma patients was lower in SKP2 protein positive expression group than that in negative expression group （P1=0.003/0.002; r=-0.275, P2=0.005）. Conclusion. The positive expression of SKP2 protein is higher in lung carcinoma than in lung benign lesion tissues, in particular, much higher in small cell lung carcinoma. In lung carcinoma, its expressive level was closely related to cell differentiation, lymph node metastasis and pathological types. Moreover, it may be an independent factor to prognosis of patients with lung carcinoma.
文摘This study describes the development of a universal phosphorylated peptide-binding protein designed to simultaneously detect serine, threonine and tyrosine kinases. The Escherichia
文摘染色体免疫共沉淀测序(Chromatin immunoprecipitation followed by sequencing,ChIP-seq)是研究DNA-蛋白质互作的有力工具,被广泛用于RNA聚合酶、转录因子和组蛋白修饰等在基因组上的精确定位。近年来,在ChIP-seq技术的基础上,科学家提出了一系列研究DNA-蛋白质互作的技术方法,提高了测序分辨率,降低了实验成本,极大推动了表观基因组学的发展。本文综述了多种DNA-蛋白质互作研究技术的原理及其应用场景,介绍了在单细胞水平上研究DNA-蛋白质互作的实现方法,并展望其未来发展的方向。
文摘Objective： To study the expressive characteristics of SKP2 protein in non-small cell lung carcinoma and it is affection to NSCLC patients＇ prognosis. Methods： The expression of SKP2 protein was detected in 89 NSCLC, 5 benign lung neoplasmas, 5 normal bronchus and lung tissues by Tissue Chip and immunohistochemistry technology. Results： The positive rate of SKP2 protein staining was （23.52±13.57）% in NSCLC tissues, significantly higher than that in benign lung neoplasmas, normal brochus and lung tissues （2.91±1.27）% （P=0.0000〈0.001）. The expressive level of SKP2 protein in NSCLC tissues was closely related to cell differentiation （PI=0.000〈0.001）, but not to age, sex, smoking history, pathological type, site, size, lymph node metastasis and TNM stage （each Pl〉0.05）. The survival analysis displayed that the NSCLC patients＇ 5 years survival rate was lower in positive expression group than that in negative expression group （P1=0.042/0.031〈0.05; r=-0.186, P2=0.000〈0.001）. Conclusion： The positive expression of SKP2 protein may play an enhancement role in the occurrence and development of NSCLC. Moreover, it may be a bad indicator to NSCLC patients＇ prognosis.
文摘目的基于基因芯片数据分析PHD锌指蛋白19(PHD finger protein 19,PHF19)在非小细胞肺癌(non-small cell lung cancer,NSCLC)中的表达及预后价值。方法下载癌症基因组图谱(The Cancer Genome Atlas,TCGA)肺癌患者数据并分析PHF19在肺癌中表达,通过基因表达数据库(Gene Expression Omnibus,GEO)下载数据集GSE30219,GSE50081筛选患者分别纳入训练集和验证集,并分析PHF19表达与肺癌患者性别、年龄、肿瘤临床分期、病理类型与无病生存率(disease free survival,DFS)、总生存率(overall survival,OS)的关系,通过在线数据库对肺癌患者PHF19及共表达相关基因进行基因本体论与京都基因和基因组百科全书(Gene Ontology and Kyoto Encyclopedia of Genes and Genomes,GO-KEGG)富集分析、免疫浸润分析。结果TCGA、GEO数据显示PHF19在肺癌中高表达(P<0.001),PHF19表达与肿瘤分期相关。PHF19低表达组的NSCLC患者较高表达组具有更长的DFS及OS(P均<0.05)。多因素COX回归分析显示PHF19为NSCLC患者独立预后影响因素(P<0.05)。绘制列线图预测肺癌患者的生存率并验证C指数表明模型准确度良好。基因富集分析显示PHF19高表达主要与细胞周期、细胞核、染色质等相关。免疫浸润分析显示PHF19与免疫细胞浸润密切相关。结论PHF19可作为预测NSCLC预后的指标,PHF19高表达是NSCLC不良预后的独立预测因子,可能是其治疗的新的靶点。
