METHODS DEVELOPMENT FOR SEQUENCING USING AN ION/TRAP

使用离子/陷阱进行测序的方法开发

• 批准号: 2187057
• 负责人: David M. Lubman
• 金额: $ 9.62万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-12 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2187057
• 关键词: biomedical equipment development; chemical association; gel electrophoresis; mass spectrometry; method development; peptides; posttranslational modifications; protein sequence

DESCRIPTION: (Adapted from the applicant's abstract) It is proposed to develop new instrumentation and methodology which can be used for sequencing peptides and post-translationally modified peptides at the low picomole level and below. This work will be developed specifically for sequencing tumor related proteins isolated using 2-D Gel electrophoresis in the 2-D Gel Laboratory at the University of Michigan. These proteins are enzymatically cleaved into peptide fractions and the resulting peptides often can not be readily sequenced by the Edman method due to either N-terminal blockage, low amounts of sample, or the inability to detect post-translational modifications. This proposal will thus involve further development of an ion trap/reflectron time-of- flight mass spectrometer and the development of various capabilities that result from the combination of this hybrid instrument. This device will be used to obtain sequence information on peptide digests using matrix-assisted laser desorption (MALDI) for producing large ions in the trap directly or by using electrospray ionization with external injection into the trap. The ion trap will act as a front end storage device for a time-of-flight mass spectrometer with capabilities for long term storage, thus providing enhanced sensitivity for detection of the ultra low levels of peptides obtained from 2-D Gels. The storage of the IT/re TOF will also provide the ability to investigate photodissociation and collision induced dissociation with MS/MS capabilities.The long term storage of large ions in the trap will be shown to be especially important for detection of long-lived unimolecular fragmentation of these large species, which has been shown to occur on a time scale beyond the capabilities of most mass spectrometers. The various factors that allow sufficient fragmentation for interpretation of the structure of peptides in this process will be investigated. In addition, the trapping capabilities of the IT/reTOF will provide sufficient resolution to resolve the isotopic distribution of the molecular ions of peptides and of their fragment peaks so that different fragments obtained from electrospray ionization can be identified according to their charge states. Ultimately, through using fragmentation patterns observed with a reflectron time-of-flight device, a demonstration of the ability to obtain structural analysis will be presented. In addition, various methods for on-line introduction of separated peptide fractions will be investigated using the enhanced resolution and speed of the IT/reTOF.

描述：(改编自申请人的摘要)建议 开发新的仪器和方法，可用于 对多肽和翻译后修饰的多肽进行测序 低皮摩尔水平及以下。这项工作将专门开发 二维凝胶分离的肿瘤相关蛋白的测序 密歇根大学二维凝胶实验室的电泳图。 这些蛋白质在酶作用下被切割成多肽部分，而 所产生的多肽通常不能很容易地被Edman测序 方法，因为N端阻塞、样本量少或 无法检测到翻译后的修改。这项提议将 因此涉及进一步开发离子陷阱/反射时间-时间-时间 飞行质谱仪及其各种性能的发展 这是这种混合乐器组合的结果。这个设备 将被用来获得关于肽的序列信息 基质辅助激光脱附(MALDI)产生大离子的研究 直接捕获或使用电喷雾电离与外部 注入陷阱中。离子陷阱将作为前端存储设备 用于长时间飞行时间质谱仪的装置 术语存储，从而为检测 从2-D凝胶中获得超低水平的多肽。数据的存储 IT/Re TOF还将提供研究光解离的能力 和碰撞引起的解离与MS/MS能力。长期 陷阱中大离子的存储将被证明是特别重要的 对检测长寿命单分子碎裂的重要意义 这些大型物种，已经被证明是在时间尺度上发生的 超过了大多数质谱仪的能力。各种因素 允许足够的碎片化来解释结构 将对这一过程中的多肽进行研究。此外， IT/reTOF的陷阱功能将提供足够的解决方案 解析肽分子离子的同位素分布 以及它们的碎片峰，以便从 电喷雾电离可以根据它们的电荷来识别 各州。最终，通过使用与 一种反射式飞行时间装置，展示了 将介绍获得结构分析的方法。此外，各种 在线介绍分离的多肽部分的方法将是 已使用IT/reTOF的增强分辨率和速度进行调查。