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11. 发明申请

US20110269161A1 Methods, Compositions and Kits for High Throughput Kinase Activity Screening Using Mass Spectrometry and Stable Isotopes 审中-公开
标题翻译：使用质谱和稳定同位素进行高通量激酶活性筛选的方法，组合物和试剂盒
公开(公告)号：US20110269161A1
公开(公告)日：2011-11-03
申请号：US13078203
申请日：2011-04-01
申请人： Steven P. Gygi , Kazuishi Kubota , Judit Villen , Yonghao Yu
发明人： Steven P. Gygi , Kazuishi Kubota , Judit Villen , Yonghao Yu
IPC分类号： C12Q1/48 , C07K7/08
CPC分类号： C12Q1/485 , G01N33/6842 , G01N33/6848
摘要： A mass-spectrometry-based method and substrates are provided herein for large scale kinome activity profiling directly from crude lysates using 90 chemically synthesized peptide substrates with amino acid sequences derived from known phosphoproteins. Quantification of peptide phosphorylation rates was achieved via the use of stable isotope labeled synthetic peptides. Half of these peptides immediately or rapidly showed robust and site-specific phosphorylation after incubation with serum-starved HEK293 cell lysate. A method and substrates for obtaining 90 simultaneous activity measurements in a single-reaction format were developed and validated. Activating kinase pathways through insulin or EGF stimulation reproducibly altered the phosphorylation rates of peptides derived from known pathway protein substrates. While examining cell-cycle-specific activities with the panel, a peptide derived from phosphoinositide 3-kinase regulatory subunit demonstrated mitotic and tyrosine-specific phosphorylation, which was confirmed to be a Src kinase site in vivo. The kinome activity profiling strategy was successfully applied with lysates of each of: cells manipulated by various combination of mitogen stimulation, pharmacological perturbation and siRNA-directed kinase knockdown; seven different breast cancer cell lines treated with gefitinib; and each of normal and cancerous tissue samples from renal cell carcinoma patients. This method concurrently measures multiple peptide phosphorylation rates to provide a diagnostic fingerprint pattern for activated kinases, protein phosphatases, modulators of these enzymes, and pathways (kinome) from as little starting material as a few cells.
摘要翻译：本文提供基于质谱法的方法和底物，用于使用90个具有源自已知磷蛋白的氨基酸序列的化学合成的肽底物直接从粗裂解物进行大规模活性物质分析。通过使用稳定同位素标记的合成肽来实现肽磷酸化速率的定量。与血清饥饿的HEK293细胞裂解物孵育后，这些肽中的一半立即或快速显示出强壮的和位点特异性磷酸化。开发并验证了用于以单反应形式获得90次同时活动测量的方法和底物。通过胰岛素或EGF刺激激活激酶途径可重复地改变衍生自已知途径蛋白底物的肽的磷酸化速率。在使用小组检查细胞周期特异性活性的同时，衍生自磷酸肌醇3-激酶调节亚基的肽表现出有丝分裂和酪氨酸特异性磷酸化，这被证实是体内的Src激酶位点。激酶活性分析策略成功应用于以下各项的裂解物：由促分裂原刺激，药理学扰动和siRNA定向激酶敲低的各种组合操纵的细胞; 用吉非替尼治疗的七种不同的乳腺癌细胞系; 以及来自肾细胞癌患者的正常和癌组织样本。该方法同时测量多肽磷酸化速率，以提供活化激酶，蛋白磷酸酶，这些酶的调节剂的诊断指纹图谱，以及与几个细胞一样少的起始材料的途径（kinome）。

12. 发明申请

US20090053742A1 Capture and release based isotope tagged peptides and methods for using the same 审中-公开
标题翻译：捕获和释放基于同位素标记的肽及其使用方法
公开(公告)号：US20090053742A1
公开(公告)日：2009-02-26
申请号：US11985768
申请日：2007-11-16
申请人： Steven P. Gygi , Scott Anthony Gerber , Carlos Augusto Gartner
发明人： Steven P. Gygi , Scott Anthony Gerber , Carlos Augusto Gartner
IPC分类号： G01N33/543 , G01N33/574
CPC分类号： G01N33/6848 , G01N33/6842 , G01N2458/15 , Y10T436/24
摘要： The invention provides non-affinity based isotope tagged peptides, chemistries for making these peptides, and methods for using these peptides. In one aspect, tags comprise a reactive site (RS) for reacting with a molecule on a protein to form a stable association with the peptide (e.g., a covalent bond) and an anchoring site (AS) group for reversibly or removably anchoring the tag to a solid phase such as a resin support. Anchoring may be direct or indirect (e.g., through a linker molecule). Preferably, the anchoring site comprises a biotin compound. Preferably, the tag comprises a mass-altering label, such as a stable isotope, such that association of the tag with the peptide can be monitored by mass spectrometry. The reagents can be used for rapid and quantitative analysis of proteins or protein function in mixtures of proteins.
摘要翻译：本发明提供了基于非亲和力的同位素标记的肽，用于制备这些肽的化学物质和使用这些肽的方法。在一个方面，标签包含用于与蛋白质上的分子反应以与肽（例如共价键）和锚定位点（AS）组稳定结合的反应位点（RS），用于可逆地或可拆卸地锚定标签固相如树脂载体。锚定可以是直接的或间接的（例如通过连接体分子）。优选地，锚定位点包含生物素化合物。优选地，标签包含质量变化标签，例如稳定同位素，使得可以通过质谱法监测标签与肽的缔合。这些试剂可用于蛋白质混合物中蛋白质或蛋白质功能的快速和定量分析。

13. 发明授权

US06989129B2 Automated capillary liquid chromatography small volume analysis system 失效
公开(公告)号：US06989129B2
公开(公告)日：2006-01-24
申请号：US10115692
申请日：2002-04-04
申请人： Lawrence J. Licklider , Steven P. Gygi , Junmin Peng
发明人： Lawrence J. Licklider , Steven P. Gygi , Junmin Peng
IPC分类号： G01N30/02 , G01N30/08
CPC分类号： G01N30/08 , G01N30/16 , G01N30/24 , G01N30/466 , G01N30/7266 , G01N2030/085 , G01N2030/162 , G01N2030/201 , G01N2030/202 , G01N2030/285 , Y10T436/24
摘要： A system for automatically performing liquid chromatography analysis of low volume liquid chemical samples at nanosecond flow rates using an analysis column that integrates a pre-concentration trapping column and a chromatography separation column terminating at an electrospray nozzle of an online mass spectrometer. The analysis column consists of a capillary having an inside diameter of between 75 and 125 microns packed throughout with a porous bed of micron particles. A branch outlet positioned 10 to 16 centimeters upstream from the nozzle divides the analysis column into an upstream pre-concentration trap and a downstream separation column. An autosampler delivers low volume liquid samples to the upstream inlet via a two-position valve. Feed connections couple the autosampler to upstream inlet when the valve is open to inject a liquid sample into the pre-concentration trap at a maximum loading flow rate in the range from 0.5 to 50 microliters/minute. Thereafter, when the valve closes, it terminates the further injection the sample, and a concentrated portion of the sample then passes though the chromatography separation column at a much slower flow rate between 10 and 1,000 nanoliters per minute. Throughput can be doubled by coupling two such analysis columns to a single autosampler using a ten-port, two position valve. A single column can be supplied through a six port two-position valve.

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