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    • 4. 发明申请
    • Inverse labeling method for the rapid identification of marker/target proteins
    • 用于快速鉴定标记/靶蛋白的逆标记方法
    • US20040106150A1
    • 2004-06-03
    • US10412964
    • 2003-04-14
    • Yingqi Karen Wang
    • G01N033/53
    • G01N33/60G01N33/58G01N33/6803G01N33/6842G01N33/6848G01N2223/6265G01N2458/15
    • A novel procedure for performing protein labeling for comparative proteomics termed inverse labeling is provided for the rapid identification of marker or target proteins. With this method, to evaluate protein expression of a disease or a drug treated sample in comparison with a control sample, two converse collaborative labeling experiments are performed in parallel. In one experiment the perturbed sample (by disease or by drug treatment) is isotopically heavy-labeled, whereas, the control is isotopically heavy-labeled in the second experiment. When mixed and analyzed with its unlabeled or isotope light counterpart for differential comparison, a characteristic inverse labeling pattern is observed between the two parallel analyses for proteins that are differentially-expressed to an appreciable level. In particularly useful embodiments, protein labeling is achieved through proteolytic 18O-incorporation into peptides as a result of proteolysis performed in 18O-water, metabolic incorporation of 15N (or 13C and 2H) into proteins, and chemically tagging proteins with an isotope-coded tag reagent such as an isotope-coded affinity tag reagent. Also provided is a novel procedure for preparing and purifying peptides from a protein solution and a novel procedure for identifying marker or target proteins, particular phosphorylated proteins, which combines the procedure for preparing and purifying peptides from a protein solution with inverse labeling.
    • 提供了用于比较蛋白质组学的蛋白质标记进行称为反向标记的新方法,用于快速鉴定标记或靶蛋白。 利用该方法,与对照样品相比较,为了评估疾病或药物处理样品的蛋白质表达,并行进行两次相反的协同标记实验。 在一个实验中,扰动样品(通过疾病或通过药物治疗)是同位素重标记的,而在第二个实验中,对照是同位素重标记的。 当与其未标记或同位素光对应物进行混合和分析以进行差异比较时,在差异表达到可感知水平的蛋白质的两个平行分析之间观察到特征反向标记模式。 在特别有用的实施方案中,蛋白质标记通过蛋白水解引入到肽中,作为在水中进行的蛋白水解,15 N(或13 C和< H)转化成蛋白质,并用同位素编码的标签试剂如同位素编码的亲和标签试剂化学标记蛋白质。 还提供了用于从蛋白质溶液制备和纯化肽的新方法和用于鉴定标记或靶蛋白的新方法,特定的磷酸化蛋白,其将从蛋白质溶液制备和纯化肽的方法与反向标记相结合。