PROTEIN COMPLEX IDENTIFICATION, MICROCHARACTERIZATION, & LIPID PROFILING

蛋白质复合物鉴定、微观表征、

• 批准号: 7796820
• 负责人: TODD D WILLIAMS
• 金额: $ 24.29万
• 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/7796820
• 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， 蛋白质在蛋白质复合物的重要生物学背景下。我们建议采用高灵敏度， 通过质谱法进行高动态范围蛋白质表征以鉴定蛋白质、检测 翻译后修饰和定量相对和绝对蛋白质水平。对此的一个假设 组织或细胞培养样品中蛋白质丰度的定量测量将揭示 目标蛋白的作用。此外，复合物位于细胞-细胞或细胞器-细胞器界面， 因此是膜微区的一部分。这些区域中的脂质组织蛋白质，作为信号 储库，捐赠或捕获活性氧产物，并密切参与的功能， 蛋白质第二个假设是，随着年龄的增长，微域的脂质谱的差异，或 操纵，将揭示蛋白质脂质关系的细节。目的1）鉴定蛋白质中分离的蛋白质 a）使用MALDI TOP和TOF/TOF来快速确认 蛋白质的ID和指导样品制备，B）在LTQ-FT上使用LC/MS/MS以改进蛋白质 毛细管HPLC和nanoUPLC鉴别覆盖率。c）改进LC/MS/MS的信息内容 使用MALDI搜索引擎对LC/MS 1“调查”数据进行LTQ-FT或QTOF仪器实验，d） 开发替代蛋白质ID验证策略。目的2）定量化学计量和检测后 a）在LTQ-FT或QTOF上使用LC/MS/MS以延伸蛋白质， 覆盖率并找到修饰的肽。B）在LTQ-FT或QTOF上使用LCMS，通过SILAC，16 O/18 O进行定量 c）改进LC/MS的数据处理。目的3）分析筏或 a）使用头柱色谱法分析鞘脂和磷脂， 组特异性MS/MS扫描，B）改进鞘脂谱分析中的定量和数据处理。我们 我希望利用特征识别与衰老过程和氧化应激最相关的蛋白质/脂质， 选择算法此外，我们的脂质和蛋白质数据将用于系统生物学背景下， 识别受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。