PROTEIN COMPLEX IDENTIFICATION, MICROCHARACTERIZATION, & LIPID PROFILING

蛋白质复合物鉴定、微观表征、

• 批准号: 8037700
• 负责人: TODD D WILLIAMS
• 金额: $ 33万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 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/8037700
• 关键词: Affect; Affinity; Age; Aging; Aging-Related Process; Algorithms; Amino Acid Sequence; Amino Acids; Biological; Blood capillaries; Caliber; Cell Culture Techniques; Cells; Cholesterol; Complex; Data; Detection; Fourier transform ion cyclotron resonance; Glean; Harvest; Head; High Pressure Liquid Chromatography; Hybrids; Ions; Label; Lipids; Maps; Mass Spectrum Analysis; Measurement; Measures; Membrane; Membrane Microdomains; Membrane Proteins; Modification; Organelles; Oxidative Stress; Pathway interactions; Peptide Sequence Determination; Peptide Signal Sequences; Peptides; Phospholipids; Post-Translational Protein Processing; Preparation; Proteins; Proteomics; Reactive Oxygen Species; Relative (related person); Role; Sampling; Scanning; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sphingolipids; Stable Isotope Labeling; Surveys; Systems Biology; Time; Tissues; Validation; base; capillary; improved; instrument; liquid chromatography mass spectrometry; mass spectrometer; protein complex; protein function; research study; stoichiometry; tool

Recent advances in proteomics have provided tools for protein characterization that allow sampling of proteins in the important biological context of protein complexes. We propose to apply high sensitivity and high dynamic range protein characterization by mass spectrometry to identify proteins, detect posttranslational modifications and quantify relative and absolute protein levels. A hypothesis of this proposal is that quantitative measurements of protein abundances in tissue or cell culture samples will reveal roles of the target proteins. Further, the complexes are at the cell-cell or organelle-organelle interface and thus are part of a membrane micro domain. The lipids in these domains organize proteins, serve as signal reservoirs, donate or trap reactive oxygen species products and are intimately involved with the function of the proteins. A second hypothesis is that the difference in lipid profile of the micro domains with age, or manipulation, will reveal details of the protein lipid relationship. Aim 1) to identify proteins isolated in protein complexes obtained with immuno- or affinity extractions, a) Use MALDI TOP and TOF/TOF to rapidly confirm the ID of proteins and guide sample preparations, b) Use LC/MS/MS on the LTQ-FT to improve protein identification coverage with capillary HPLC and nanoUPLC. c) Improve information content from LC/MS/MS experiments on the LTQ-FT or QTOF instruments using MALDI search engines on LC/MS1 "survey" data, d) Develop an alternative protein ID validation strategy. Aim 2) To quantify stoichiometry and detect post translational modifications in protein complexes, a) Use LC/MS/MS on LTQ-FT or QTOF to extend protein coverage and find modified peptides. b) Use LCMS on LTQ-FT or QTOF for quantitation by SILAC, 16O/18O labeling, or spiked internal standards c) Improve data handling from LC/MS. Aim 3) Profile lipids in raft or membrane preparations from membranes at different ages, a) Profile sphingo- and phospholipids using head group specific MS/MS scans, b) Improve the quantitation and data handling in sphingolipid profiling. We hope to identify proteins/lipids that are most relevant to the aging process and oxidative stress using feature selection algorithms. In addition, our lipid and protein data will be used in a systems biology context to identify biological networks and pathways affected by aging and oxidative stress via mapping the proteins/lipids to known pathways.

蛋白质组学的最新进展为蛋白质表征提供了工具，允许对 蛋白质在蛋白质复合物的重要生物学环境中的蛋白质。我们建议应用高灵敏度， 高动态范围蛋白质通过质谱法表征以鉴定蛋白质，检测 翻译后修饰并量化相对和绝对蛋白水平。一个假设 建议是，将揭示组织或细胞培养样品中蛋白质丰度的定量测量 靶蛋白的作用。此外，这些复合物位于细胞细胞或细胞器 - 轨道界面， 因此是膜微结构域的一部分。这些结构域中的脂质组织蛋白质，用作信号 储层，捐赠或陷阱活性氧产品，并与 蛋白质。第二个假设是，随着年龄的年龄或 操纵将揭示蛋白质脂质关系的细节。目标1）鉴定在蛋白质中分离的蛋白质 通过免疫或亲和力提取获得的复合物，a）使用MALDI顶部和TOF/TOF快速确认 蛋白质的ID和引导样品制剂，b）使用LTQ-FT上的LC/MS/MS来改善蛋白质 毛细管HPLC和NanouPlc的识别覆盖范围。 c）从LC/MS/MS改善信息内容 使用MALDI搜索引擎在LC/MS1“调查”数据上进行LTQ-FT或QTOF仪器的实验，D） 制定替代蛋白质ID验证策略。目标2）量化化学计量和检测柱 蛋白质复合物中的翻译修饰，a）在LTQ-ft或QTOF上使用LC/MS/MS扩展蛋白质 覆盖范围并找到改良的肽。 b）在LTQ-FT或QTOF上使用LCMS进行定量，Silac，16o/18o 标记或尖刺的内部标准c）从LC/MS改善数据处理。目标3）木筏或 来自不同年龄的膜的膜制剂，a）使用头部的特征丛和磷脂。 组特定的MS/MS扫描，b）改进鞘脂分析中的定量和数据处理。我们 希望鉴定与衰老过程和使用特征最相关的蛋白质/脂质 选择算法。此外，我们的脂质和蛋白质数据将在系统生物学环境中使用 通过映射衰老和氧化应激影响的生物网络和途径 蛋白质/脂质到已知途径。