Mass Spectrometry Core

质谱核心

• 批准号: 7225436
• 负责人: LEWIS C. CANTLEY
• 金额: $ 15.77万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-12-01 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7225436
• 关键词: Affect; Amines; Amino Acids; Binding Proteins; Bioinformatics; Biological; Budgets; Cells; Complex; Coupled; Culture Media; Cultured Cells; Development; Digestion; Dissection; Drosophila genus; Epitopes; Gel; Genes; Housing; Human; Individual; Ions; Label; Light; Liquid Chromatography; Maps; Mass Spectrum Analysis; Methods; Modification; Pathogenesis; Pathway interactions; Peptides; Peutz-Jeghers Syndrome; Phase; Phosphopeptides; Phosphorylation; Phosphorylation Site; Preparation; Principal Investigator; Protein Analysis; Protein Binding; Protein Databases; Protein Kinase; Proteins; Proteolysis; Proteomics; Published Comment; Pulsar; Reagent; Relative (related person); Research; Research Design; Resources; Role; Sampling; Services; Signal Pathway; Signal Transduction; Site; Stable Isotope Labeling; Standards of Weights and Measures; TSC1 gene; TSC1/2 gene; TSC2 gene; Technology; Time; Tuberous Sclerosis; Yeasts; cell growth; data management; data mining; human FRAP1 protein; in vivo; mass spectrometer; programs; protein aminoacid sequence; protein protein interaction; research study; software development; stable isotope; tandem mass spectrometry

The Mass Spectrometry and Proteomics Core will provide protein identification, phosphorylation site mapping, and develop strategies for relative quantification of proteins/phosphopeptides in order to aid in the understanding of the Tuberous sclerosis pathway and pathogenesis, LKB1/AMPK signaling and its role in Peutz-Jeghers syndrome, and a dissection of Tsc1/Tsc2/Tor/S6K signaling. Liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) will be used to identify proteins and phosphorylation sites as well as provide quantitative information for proteins and phosphorylation sites. Peptides from tryptic digestions will be analyzed by microcapillary reversed-phase LC/MS/MS using a LTQ linear ion trap mass spectrometer and a QSTAR Pulsar quadrupole-TOF mass spectrometer. Proteins and their phosphorylation sites will be identified after gel separation by interrogating non-redundant protein databases with peptide tandem mass spectra. Relative quantitation of proteins will be performed by isotopically labeling the proteins from different cell states with light and heavy amino acids, respectively, using stable isotope labeling of amino acids in cell culture (SILAC) using the QSTAR mass spectrometer or labeling post cell growth at the protein and/or peptide level using global internal standard technology (GIST) primary amine stable isotope labeling reagents. Ratios of light to heavy peptide pairs will be determined using both MSQuant and in-house developed software. Bioinformatics will be used for data management and data mining for biological significance.

质谱和蛋白质组学核心将提供蛋白质鉴定，磷酸化位点， 绘图，并制定蛋白质/磷酸肽相对定量的策略，以帮助 了解脑硬化的途径和发病机制，LKB 1/AMPK信号传导及其在 Peutz-Jeghers综合征和Tsc 1/Tsc 2/Tor/S6 K信号传导的解剖。液相色谱 串联质谱（LC/MS/MS）将用于鉴定蛋白质和磷酸化位点 为蛋白质和磷酸化位点提供定量信息。胰蛋白酶消化肽 将使用LTQ线性离子阱质谱仪通过微毛细管反相LC/MS/MS进行分析 和QSTAR脉冲星四极飞行时间质谱仪蛋白质及其磷酸化位点将被 凝胶分离后通过用肽串联质量查询非冗余蛋白质数据库进行鉴定 谱蛋白质的相对定量将通过同位素标记来自不同来源的蛋白质来进行。 利用细胞内氨基酸的稳定同位素标记， 使用QSTAR质谱仪或在蛋白质处标记细胞生长后的细胞培养物（SILAC），和/或 肽水平使用全球内标技术（GIST）伯胺稳定同位素标记 试剂将使用MSQuant和内部测定轻肽与重肽对的比率 开发软件。生物信息学将用于生物信息学的数据管理和数据挖掘。 意义