PROTEIN COMPLEX IDENTIFICATION, MICROCHARACTERIZATION, & LIPID PROFILING

蛋白质组学的最新进展为蛋白质表征提供了工具，允许对 蛋白质是蛋白质复合体中重要的生物背景。我们建议应用高灵敏度和 高动态范围蛋白质表征的质谱学鉴定蛋白质，检测 翻译后修饰并量化相对和绝对蛋白质水平。关于这一点的假设 建议对组织或细胞培养样本中蛋白质丰度的定量测量将揭示 靶蛋白的作用。此外，复合体位于细胞-细胞或细胞器-细胞器界面，并且 因此是膜微域的一部分。这些区域中的脂类组织蛋白质，作为信号 水库，捐赠或捕获活性氧物种的产物，并密切参与 蛋白质。第二种假设是微域的脂质分布随年龄的不同而不同，或 操纵，将揭示蛋白质与脂质关系的细节。目的1)鉴定分离于蛋白质中的蛋白质 通过免疫或亲和提取获得的络合物，a)使用MALDI TOP和TOF/TOF快速确认 B)利用LC/MS/MS对LTQ-FT上的蛋白质进行改进 毛细管高效液相色谱和纳米超高效液相色谱鉴定覆盖率。C)提高LC/MS/MS的信息含量 使用MALDI搜索引擎在LTQ-FT或QTOF仪器上对LC/MS1“调查”数据进行的实验，d) 开发一种替代的蛋白质ID验证策略。目标2)量化化学计量和检测POST 蛋白质复合体的翻译修饰，a)利用LTQ-FT或QTOF上的LC/MS/MS延伸蛋白质 覆盖并找到修饰的多肽。B)在LTQ-FT或QTOF上使用LC MS，以供SILAC定量，16O/18O 标签或添加的内标c)改进LC/MS的数据处理目的3)RAFT或 由不同年龄的膜制备的膜，a)使用Head的轮廓神经鞘氨醇和磷脂 针对特定群体的MS/MS扫描，b)改进鞘脂分析中的定量和数据处理。我们 希望使用功能识别与衰老过程和氧化应激最相关的蛋白质/脂类 选择算法。此外，我们的脂肪和蛋白质数据将用于系统生物学背景下 通过绘制受衰老和氧化应激影响的生物网络和途径图，确定生物网络和途径 蛋白质/脂类通向已知的途径。

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.