UNMC: PROTEOMICS

UNMC：蛋白质组学

• 批准号: 8167510
• 负责人: PAWEL S CIBOROWSKI
• 金额: $ 5.86万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8167510
• 关键词: Acetylation; Actins; Affinity Chromatography; Biological Markers; Cells; Characteristics; Complex Mixtures; Computer Retrieval of Information on Scientific Projects Database; Custom; Data Analyses; Detection; Detergents; Excision; Fluorescent Dyes; Fractionation; Funding; Future; Goals; Grant; Individual; Institution; Isoelectric Point; Label; Liquid Chromatography; Mass Spectrum Analysis; Methods; Molecular Weight; N-terminal; Outcome; Peptide Mapping; Peptides; Property; Protein Isoforms; Proteins; Proteomics; Protocols documentation; Research; Research Personnel; Resources; Sampling; Screening procedure; Source; Spottings; System; Techniques; Technology; United States National Institutes of Health; Work; base; cyanine dye 5; design; experience; high throughput screening; instrument; ionization; mass spectrometer; protein aminoacid sequence; protein expression; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. It is of great importance for the productive outcome that all proteomics technologies are integrated in a logical way leading from the identification of unique differences in protein expression (biomarkers) in a complex mixture to the identification and full characterization of a protein in question. It is inevitable that each approach has strengths and weaknesses which will be considered in designing work-flow protocols. For example, every study will begin with at least one-dimensional fractionation to reduce sample complexity before screening and/or profiling can be applied. This is very important step because of two reasons. One is to reduce usually high range of concentration of proteins (high v. low abundant) in sample. Second, mass spectrometric analyses based on MALDI-TOF mode of protein/peptide ionization are very sensitive to contamination with detergents. Another example is to use custom design affinity chromatography columns. Removal of actin from cell lysates will vastly reduce the complexity of an initial sample. Another goal is to integrate high throughput assays with in-depth analyses. From our perspective 2DE is very useful technique because of its ability to make targeted analysis of selected protein spots instead of analyzing entire sample containing proteins, which are differentially expressed mixed with those which are not. Detection of proteins labeled with different fluorescent dyes in 2DE DIGE (Cy3, green and Cy5, red) greatly enhances analytical power of this technique of profiling because it does not reduce the ability of protein identification. Sensitivity of established method of protein identification based on peptide fingerprinting using ABI4800 MALDI-TOF/TOF instrument is approximately 10 fmoles. This approach is also a high throughput one. Moreover, isoforms of proteins which are different in PTMs such as N-terminal acetylation can be separated by 2DE, because they will differ in isoelectric point, but their difference in molecular weight or physicochemical properties are too small to separate them by other means such as 1DE or liquid chromatography. ABI4800 mass spectrometer also allows peptide sequencing in TOF/TOF configuration if enough material is available in the sample already used for peptide fingerprinting. Our experience indicates that 50 fmoles of peptide mixture in one sample can be used for 3 to 4 analyses. This enable us to perform one peptide fingerprinting determination in a TOF configuration and 2 to 3 individual peptides can be subsequently sequenced. Although 2DE DIGE is a powerful profiling technique, mass spectrometry based techniques such as iTRAQ offer additional information. It is now quite obvious that one profiling approach will reveal only partial characteristics of an investigated sample. In our future experiments we will use two or more techniques. Our current effort is directed to create a comprehensive system of data analysis.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 对于生产结果而言，所有蛋白质组学技术都以逻辑方式整合起来，从而从复杂的混合物中识别蛋白质表达（生物标志物）的唯一差异到所讨论的蛋白质的鉴定和充分表征，这一点至关重要。 每种方法都有优势和劣势是不可避免的，这些优点和劣势将在设计工作流程方案时被考虑。 例如，每项研究都将从至少一维分馏开始，以在筛选和/或分析之前降低样品复杂性。 这是非常重要的一步，因为有两个原因。 一种是降低样品中通常高的蛋白质浓度（高v。低量）。 其次，基于蛋白质/肽电离模式的质谱分析对对洗涤剂的污染非常敏感。 另一个示例是使用自定义设计亲和力色谱列。 从细胞裂解物中去除肌动蛋白将大大降低初始样品的复杂性。 另一个目标是将高吞吐量测定与深入分析相结合。 从我们的角度来看，2DE是非常有用的技术，因为它能够对所选蛋白质斑点进行靶向分析，而不是分析包含蛋白质的整个样品，这些蛋白质与未与那些的蛋白质差异表达。在2DE DIGE（CY3，Green和Cy5，Red）中用不同荧光染料标记的蛋白质检测大大提高了这种分析技术的分析能力，因为它不会降低蛋白质鉴定的能力。基于ABI4800 MALDI-TOF/TOF仪器基于肽指纹识别的蛋白质识别方法的敏感性约为10 fmoles。 这种方法也是一种高吞吐量。此外，在PTM中不同的蛋白质（例如N末端乙酰化）的同工型可以通过2D分离，因为它们在等电点上会有所不同，但是它们在分子量或物理化学特性方面的差异太小，无法用其他方式分离它们，例如1DE或液态色谱。如果已经用于肽指纹的样品中有足够的材料，则ABI4800质谱仪还可以在TOF/TOF构型中进行肽测序。 我们的经验表明，一个样品中的50个肽混合物可用于3至4个分析。 这使我们能够在TOF构型中执行一个肽指纹测定，随后可以对2至3个单个肽进行测序。 尽管2DE DIGE是一种强大的分析技术，但诸如ITRAQ之类的基于质谱的技术提供了其他信息。 现在很明显，一种分析方法只会揭示研究样本的部分特征。 在未来的实验中，我们将使用两种或更多技术。 我们目前的努力旨在创建一个全面的数据分析系统。