UNMC: PROTEOMICS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。次级项目的主要支助 子项目的主要研究者可能是由其他来源提供的， 包括其它NIH来源。 为子项目列出的总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 所有蛋白质组学技术以逻辑的方式整合，从复杂混合物中蛋白质表达（生物标志物）的独特差异的鉴定到所讨论蛋白质的鉴定和全面表征，对于生产性结果非常重要。 不可避免的是，每种方法都有优缺点，这将在设计工作流协议时加以考虑。 例如，每项研究将开始时至少进行一维分级，以在进行筛选和/或分析之前降低样本复杂性。 这是非常重要的一步，原因有二。 一种是降低样品中通常高范围的蛋白质浓度（高丰度与低丰度）。 其次，基于蛋白质/肽电离的MALDI-TOF模式的质谱分析对去污剂的污染非常敏感。 另一个例子是使用定制设计的亲和色谱柱。 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。 另一个目标是将高通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，因为它能够对选定的蛋白质点进行靶向分析，而不是分析含有蛋白质的整个样品，这些蛋白质与那些没有差异表达的蛋白质混合。在2DE DIGE中用不同荧光染料（Cy 3，绿色和Cy 5，红色）标记的蛋白质的检测极大地增强了这种谱分析技术的分析能力，因为它不降低蛋白质鉴定的能力。建立的基于肽指纹图谱的蛋白质鉴定方法使用ABI 4800 MALDI-TOF/TOF仪器的灵敏度约为10 fmoles。 该方法也是高通量方法。此外，可以通过2DE分离PTM不同的蛋白质的同种型，例如N-末端乙酰化，因为它们的等电点不同，但是它们的分子量或物理化学性质的差异太小，不能通过其他方法例如1DE或液相色谱法分离它们。ABI 4800质谱仪还允许在TOF/TOF配置中进行肽测序，前提是已用于肽指纹图谱分析的样品中有足够的材料。 我们的经验表明，一个样品中50 fmol的肽混合物可用于3至4次分析。 这使我们能够在TOF配置中执行一个肽指纹测定，并且随后可以对2至3个单独的肽进行测序。 虽然2DE DIGE是一种强大的分析技术，但基于质谱的技术（如iTRAQ）可提供更多信息。 现在很明显，一种特征分析方法只能揭示所调查样本的部分特征。 在我们未来的实验中，我们将使用两种或更多种技术。 我们目前的努力是建立一个全面的数据分析系统。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.