The Proteomics Core

蛋白质组学核心

• 批准号: 7864265
• 负责人: John Edward Wiktorowicz
• 金额: $ 19.51万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7864265
• 关键词: Analysis of Variance; Arts; Biological Assay; Blood Vessels; Computer software; Contracts; Cost Savings; Data; Data Analyses; Data Quality; Dependence; Dissection; Doctor of Philosophy; Ensure; Equipment; Fingerprint; Fractionation; Funding; Gel; Human; Image Analysis; Immunoassay; Individual; Label; Laboratories; Liquid Chromatography; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Measurement; Medical; Methodology; Mus; National Heart, Lung, and Blood Institute; Pattern; Peptides; Preparation; Protein Analysis; Proteins; Proteomics; Reagent; Recombinants; Reproducibility; Research Infrastructure; Resources; Running; Sampling; Services; Smooth Muscle Myocytes; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Staining method; Stains; Students; Testing; Texas; Thoracic Aortic Aneurysm; Time; Two-Dimensional Gel Electrophoresis; Universities; Validation; Variant; base; chemokine; cytokine; dosage; instrument; protein complex; protein expression; stable isotope; synthetic polymer Bioplex; tandem mass spectrometry

Core E will provide state-of-the-art protein analysis services for SCCOR projects as a high quality, centralized resource to provide consistency and reproducibility in sample preparation, data analysis, and cost savings by eliminating duplications in equipment and reagents. Core E is based in the Biomolecular Resource Facility (BRF), an established Institutional Core Laboratory of the University of Texas Medical Branch that is partially funded by the NHLBI Proteomics Center contract mechanism. The BRF occupies 6,200 ft2 distributed within twelve laboratories and seven offices located centrally in the campus at UTMB. The current scientific staff includes 17 (6 Ph.D., 3 M.S., and 6 B.S.) individuals. Specifically, the Proteomics Core will provide established methodologies for sample pre-separation fractionation, 2-dimensional gel electrophoresis (2DE), differential protein staining, gel imaging and analysis, peptide labeling with stable isotopes (iTRAQ) and quantitative mass spectrometry, protein identification by peptide mass fingerprinting via matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS), liquid chromatography tandem mass spectrometry (LC/MS/MS), and Luminex multiplex assays for determination of cytokine expression patterns. The specific objectives of the Proteomics Core are: 1. Provide the infrastructure necessary for consistent sample pre-separation fractionation (Project 1); 2. Perform differential protein expression analysis by 2DE and identification of proteins from vascular smooth muscle cells and aortic explant cultures (Projects 1 and 3); 3. Perform differential iTRAQ labeling, LC/MS/MS protein identification of complex protein samples (Projects 1 and 3); and 3. Perform immunoassays of human and mouse chemokines/cytokines in aortic explant cultures from human and mouse samples (Projects 3 and 4). To ensure data quality, validation activities using standard proteins and/ or peptides have been established. Validation of data generated by image analysis with Progenesis software is accomplished utilizing appropriate statistical analyses including Student's t-test for hypothesis and significance testing, Multiple Hypothesis testing corrections, Hierarchical Clustering of control vs. treated, and Analysis of Variance (ANOVA) for time course/dosage dependence of expression. For MS, data quality is ensured through the use of appropriate internal and external standards, while MS instruments are routinely calibrated with external standards. For the Bioplex cytokine measurements, recombinant standards are run with each plate and sample-to-sample variation determined.

核心E将为SCCOR项目提供最先进的蛋白质分析服务，作为高质量的集中式 资源可在样本准备，数据分析和节省成本的一致性和可重复性中提供 消除设备和试剂的重复。核心E基于生物分子资源设施（BRF）， 德克萨斯大学医学分支机构已建立的机构核心实验室，部分由 NHLBI蛋白质组学中心合同机制。 BRF占有6200 ft2，分布在十二个之内 实验室和七个办公室位于UTMB校园中心。目前的科学人员包括17个（6 博士学位，3 M.S.和6 B.S.）个人。具体而言，蛋白质组学核心将提供既定的方法论 用于样品前分离分馏，二维凝胶电泳（2DE），差异蛋白染色，凝胶 成像和分析，具有稳定同位素（ITRAQ）的肽标记和定量质谱法，蛋白 通过基质辅助激光解吸电离时间通过肽质量指纹识别飞行质量的电离时间 光谱法（MALDI TOF MS），液相色谱串联质谱法（LC/MS/MS）和Luminex 多重测定方法用于测定细胞因子表达模式。蛋白质组学核心的特定目标 为：1。提供一致样本前分离分馏所必需的基础架构（项目1）； 2。 通过2DE进行鉴别蛋白表达分析，并鉴定来自血管平滑肌的蛋白质 细胞和主动脉外植体培养物（项目1和3）； 3。执行差异ITRAQ标签，LC/MS/MS蛋白 鉴定复杂蛋白质样品（项目1和3）；和3。执行人和老鼠的免疫测定 来自人类和小鼠样品的主动脉外植体培养物中的趋化因子/细胞因子（项目3和4）。确保 已经建立了使用标准蛋白和/或肽的数据质量，验证活动。验证 使用适当的统计来完成图像分析生成的数据 分析包括学生的t检验，用于假设和显着性检验，多个假设检验校正， 控制与处理的层次结构聚类，以及方差分析（ANOVA），用于时间课程/剂量 表达的依赖性。对于MS，通过使用适当的内部和外部来确保数据质量 标准标准，而MS仪器则经常用外部标准进行校准。对于Bioplex细胞因子 测量，重组标准标准，每个板和样品对样品变化都确定。