Parallel Peptide Tandem Mass Spectrometry (MS)

描述(由申请人提供)： 这项提案的R21阶段的具体目标是开发必要的软件工具，以解释由碰撞诱导解离(CID)产生的多肽的串联谱，而不是像猎枪蛋白质组学中通常采用的串联质谱学，并在从多肽标准到从髓母细胞瘤原代细胞系裂解物提取的蛋白质的日益复杂的样本上证明其优于串联CID。在傅立叶变换离子回旋共振质谱仪(FT-ICRMS)中，我们提出的技术被称为鸟枪CID和鸟枪串联质谱仪(MS/MS)，以区别于串联MS和多路MS。这一R21-R33组合方案的总体目标是为FT-ICR-MS中的多路MS的优势提供一个具有成本效益的竞争对手。我们的方法将使用分辨率和质量精度较低的飞行时间(TOF)质谱仪和连续交替的母离子测量数据采集方案，然后在样品的整个色层引入过程中进行碎片离子测量。蛋白质将通过在整个层析时间内的亲本多肽离子的质量图谱、独特的层析限制及其组合的碎片离子来识别。原发脑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的神经外胚层肿瘤，通常起源于小脑疣，表现出不同程度的神经分化受阻。在发育过程中，小脑需要内源性维甲酸来适当地控制神经元的凋亡和分化。在胚胎癌和神经母细胞瘤细胞中，维甲酸诱导神经分化和细胞周期停滞。最近研究表明，维甲酸能诱导髓母细胞瘤细胞系和新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。弗雷德·哈钦森癌症研究所的吉姆·奥尔森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第三阶段临床试验，我们将与他一起在一个模型中开发鸟枪式CID，该模型寻求：1)促进识别与髓母细胞瘤细胞系的维甲酸反应相关的标记物；2)确定维甲酸途径的关键成分，该途径受维甲酸反应性髓母细胞瘤细胞的治疗调节。这项工作将在髓母细胞瘤细胞系和来自维甲酸敏感和耐药肿瘤的原代细胞系中完成。

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.