Parallel Peptide Tandem Mass Spectrometry (MS)

描述（由申请人提供）： 这是本提案的R21阶段的具体目标是开发必要的软件工具来解释串联质谱产生的碰撞诱导解离（CID）的肽在平行，而不是在系列中，因为通常在鸟枪蛋白质组学实践，并证明其优势超过串行CID的样品越来越复杂的肽标准蛋白质提取的髓母细胞瘤原代细胞系裂解物。我们提出的技术被称为鸟枪CID和鸟枪串联质谱（MS/MS），以区别于串联串联MS和多重MS的傅里叶变换离子回旋共振质谱仪（FT-ICRMS）。 这是R21-R33组合建议的总体目标，提供一个具有成本效益的竞争对手的优势，多重MS在FT-ICR-MS。我们的方法将使用较低的分辨率和质量精度的飞行时间（TOF）质量分析仪和连续交替的数据采集方案的母离子测量随后碎片离子测量整个色谱引入样品。将通过整个色谱时间内母体肽离子的质量图谱、独特色谱限制及其组合碎片离子的组合鉴别蛋白质。 原发性脑肿瘤是儿童癌症相关死亡的主要原因。髓母细胞瘤是一种原始神经外胚层肿瘤，通常起源于小脑蚓部，表现出不同程度的神经分化停滞。小脑发育过程中需要内源性类维生素A来适当控制神经元凋亡和分化。在胚胎癌和神经母细胞瘤细胞中，类维生素A诱导神经分化和细胞周期停滞。近年来研究表明，类维生素A可诱导髓母细胞瘤细胞系和新鲜切除的髓母细胞瘤细胞发生广泛的细胞凋亡和神经元分化。Fred哈钦森癌症研究所的Jim Olson博士正在组织一项关于13-顺式维甲酸在髓母细胞瘤治疗中的作用的III期临床试验，我们将与他一起在一个模型中开发鸟枪CID，该模型旨在：1）有助于鉴定髓母细胞瘤细胞系中与类维生素A反应性相关的标志物，和2）定义视黄酸途径的关键组成部分，视黄酸反应性髓母细胞瘤细胞治疗调节。这项工作将与髓母细胞瘤细胞系和原代细胞系衍生的类维生素A敏感和耐药肿瘤。

DESCRIPTION (provided by applicant): It is the specific aim of the R21 phase of this proposal to develop the software tools necessary to interpret tandem mass spectra produced by collision induced dissociation (CID) of peptides in parallel rather than in series, as is commonly practiced in shotgun proteomics, and to prove its advantages over serial CID on samples of increasing complexity from peptide standards to proteins extracted from a medulloblastoma primary cell line lysate. Our proposed technology is referred to as shotgun CID and shotgun tandem mass spectrometry (MS/MS) to distinguish it from serial tandem MS and multiplex MS in a Fourier transform-ion cyclotron resonance-mass spectrometer (FT-ICRMS). It is the general aim of this combined R21-R33 proposal to provide a cost effective competitor to the advantages of multiplex MS in FT-ICR-MS. Our approach will use lower resolution and mass accuracy time-of-flight (TOF) mass analyzers and a continuously alternating data acquisition scheme of parent ion measurement followed fragment ion measurement throughout the chromatographic introduction of sample. Proteins will be identified by a combination of mass mapping of parent peptide ions across the entire chromatographic time, unique chromatographic constraints and their combined fragment ions. Primary brain tumors are the leading cause of cancer-related death in children. Medulloblastoma is a primitive neuroectodermal tumor that typically arises from the cerebellar vermis and shows variable degrees of arrested neural differentiation. The cerebellum requires endogenous retinoids for proper control of neuronal apoptosis and differentiation during development. In embryonal carcinoma and neuroblastoma cells, retinoids induce neural differentiation and cell-cycle arrest. Retinoids have recently been shown to induce extensive apoptosis and neuronal differentiation in medulloblastoma cell lines and freshly resected medulloblastoma cells. Together with Dr. Jim Olson of the Fred Hutchinson Cancer Research Institute, who is organizing a Phase III clinical trial on the effects of 13-cis retinoic acid in medulloblastoma therapy, we will develop shotgun CID within a model that seeks to: 1) facilitate identification of markers associated with retinoid-responsiveness in medulloblastoma cell lines and 2) define key components of the retinoic acid pathway modulated by treatment in retinoid-responsive medulloblastoma cells. This work will be done with medulloblastoma cell lines and primary cells lines derived from retinoid-sensitive and -resistant tumors.