Photocleavable ICAT Reagents for Quantitative Proteomics

描述(申请人提供)：蛋白质表达谱正在成为监测肿瘤疾病进程以及识别与该疾病相关的诊断生物标记物的最具吸引力的工具之一。虽然已经有多种蛋白质组学工具被广泛研究，包括双向凝胶电泳法和质谱仪，但由于这些方法的准确性有限且不能测量整个蛋白质组，定量蛋白质组学仍然很难实现。这个项目的目标是通过开发一种新的基于光可切割同位素编码亲和标签(PC-ICAT)和质谱学的定量蛋白质组学方法来克服这些限制。PC-ICAT试剂由四个不同的元件组成：一个生物素亲和标记、一个能快速有效地进行光切割的可光降解连接基、一个可变质量连接基和一个化学反应基团。轻质和重质PC-ICAT试剂用于标记两种不同的样本，例如正常组织和疾病相关组织的提取物。经过消化、捕获和光释放后，用LC-MS测量每个样品中标记多肽的相对丰度。在该项目的第一阶段，已经合成了一种巯基反应性PC-ICAT马来酰亚胺试剂，并使用模型肽和蛋白质对其进行了广泛的评价。值得注意的是，与传统的CAT试剂相比，PC-ICAT的性能表现出更好的性能。在第二阶段，我们将通过以下方式扩大PC-ICAT在蛋白质组学研究和临床应用中的应用：1)合成和评估PC-ICAT试剂的胺反应型；2)评估固体支撑型PC-ICAT试剂；3)探索PC-ICAT试剂在蛋白质表达谱和生物标记物发现中的应用。为此，我们将应用PC-ICAT试剂和LC-MS来研究来自正常和恶性人类前列腺细胞系的细胞裂解物。质谱学和蛋白质组学领域的专家凯西·科斯特洛博士将在该项目的第二阶段担任顾问。

DESCRIPTION (provided by applicant): Protein expression profiling is becoming one of the most attractive tools for monitoring the course of neoplastic disease as well as identifying diagnostic biomarkers associated with the disease. While several proteomic tools have been extensively investigated for this purpose, including two-dimensional gel electrophoresis and mass spectrometry, quantitative proteomics is still difficult to achieve due to the limited accuracy and inability of these methods to survey the entire proteome. The goal of this project is to overcome these limitations by developing a new approach to quantitative proteomics based on the use of PhotoCleavable Isotope Coded Affinity Tags (PC-ICAT) along with mass spectrometry. PC-ICAT reagents are composed of four distinct elements: a biotin affinity tag, a photodeavable linker which exhibits rapid and efficient photocleavage, a variable mass linker and a chemically reactive group. The light and heavy versions of the PC-ICAT reagents are used to tag two different samples, for example extracts from normal and disease related tissues. After digestion, capture and photorelease, the relative abundance of the tagged peptides in each sample is measured using LC-MS. During Phase I of this project, a sulfhydryl reactive PC-ICAT maleimide reagent has been synthesized and extensively evaluated using both model peptides and proteins. Significantly, the performance of PC-ICAT exhibits superior performance compared with the conventional (CAT reagent. During Phase II, we will extend the usefulness of PC-ICAT in proteomic research and clinical applications by: 1) synthesizing and evaluating an amine reactive version of the PC-ICAT reagent, 2) evaluating a solid supported PC-ICAT reagent and 3) exploring the use of PC-ICAT reagents for protein expression profiling and biomarker discovery. For this purpose, we will apply PC-ICAT reagents and LC-MS to study cell lysates derived from normal and malignant human prostate cell lines. Dr. Cathy Costello, an expert in the area of mass spectrometry and proteomics will serve as a consultant during Phase II of this project.