PROTEIN COMPLEX IDENTIFICATION, MICROCHARACTERIZATION, & LIPID PROFILING

蛋白质复合物鉴定、微观表征、

• 批准号: 8234021
• 负责人: TODD D WILLIAMS
• 金额: $ 16.48万
• 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/8234021
• 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; Validation; base; capillary; improved; instrument; liquid chromatography mass spectrometry; mass spectrometer; protein complex; protein function; research study; stoichiometry; tissue culture; 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，可以对蛋白质进行采样 蛋白质复合物的重要生物学背景。我们建议采用高灵敏度和 通过质谱法进行高动态范围蛋白质表征，以识别蛋白质、检测 翻译后修饰并量化相对和绝对蛋白质水平。对此的一个假设 建议对组织或细胞培养样品中的蛋白质丰度进行定量测量将揭示 靶蛋白的作用。此外，复合物位于细胞-细胞或细胞器-细胞器界面处并且 因此是膜微域的一部分。这些结构域中的脂质组织蛋白质，充当信号 储存库，捐赠或捕获活性氧产物，并与以下功能密切相关 蛋白质。第二个假设是微区的脂质谱随年龄的变化，或者 操纵，将揭示蛋白质脂质关系的细节。目标 1) 鉴定蛋白质中分离的蛋白质 通过免疫或亲和提取获得的复合物，a) 使用 MALDI TOP 和 TOF/TOF 快速确认 蛋白质的 ID 并指导样品制备，b) 在 LTQ-FT 上使用 LC/MS/MS 来改进蛋白质 毛细管 HPLC 和 nanoUPLC 的鉴定覆盖率。 c) 改进 LC/MS/MS 的信息内容 使用 MALDI 搜索引擎在 LTQ-FT 或 QTOF 仪器上对 LC/MS1“调查”数据进行实验，d) 开发替代的蛋白质 ID 验证策略。目标 2) 量化化学计量并检测后 蛋白质复合物中的翻译修饰，a) 在 LTQ-FT 或 QTOF 上使用 LC/MS/MS 来延伸蛋白质 覆盖范围并找到修饰的肽。 b) 在 LTQ-FT 或 QTOF 上使用 LCMS，通过 SILAC、16O/18O 进行定量 标签或加标内标 c) 改进 LC/MS 的数据处理。目标 3) 分析筏中的脂质或 使用不同年龄的膜制备膜，a) 使用头分析鞘磷脂和磷脂 组特异性 MS/MS 扫描，b) 改进鞘脂分析中的定量和数据处理。我们 希望利用特征来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 通过绘制衰老和氧化应激的图谱来识别受衰老和氧化应激影响的生物网络和途径 蛋白质/脂质至已知途径。