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: 2187055
• 负责人: David M. Lubman
• 金额: $ 11.32万
• 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/2187055
• 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：（改编自申请人摘要）拟 开发新的仪器和方法，可用于 测序肽和后修饰的肽， 低皮摩尔水平及以下。这项工作将专门制定 用于测序使用2-D凝胶分离的肿瘤相关蛋白 在密歇根大学的2-D凝胶实验室中进行电泳。 这些蛋白质被酶促裂解成肽级分， 得到的肽通常不能容易地通过Edman测序 方法由于N-末端阻断，样品量低，或 无法检测翻译后修饰。 这项建议会 因此涉及离子阱/反射器时间的进一步发展， 飞行质谱仪和各种能力的发展 这是由这种混合工具的组合产生的。 这个设备 将用于获得肽序列信息， 基质辅助激光解吸（MALDI），用于在 直接捕获或通过使用电喷雾电离与外部 注入陷阱。 离子阱将作为前端存储器 用于具有长时间测量能力的飞行时间质谱仪的装置 术语存储，从而提供用于检测 从2-D凝胶中获得的超低水平的肽。 的存储 IT/re TOF还将提供研究光解离的能力 和碰撞诱导解离与MS/MS能力。长期 在阱中存储大离子将被证明是特别 重要的是检测长寿命的单分子断裂 这些大型物种，已被证明发生在一个时间尺度上， 超出了大多数质谱仪的能力。 的各种因素 允许足够的碎片来解释结构 在这个过程中的肽将被调查。 此外该 IT/reTOF的捕获能力将提供足够的分辨率 解析多肽分子离子的同位素分布 以及它们的碎片峰， 电喷雾电离可以根据它们的电荷来识别 states. 最后，通过使用 一个反射器飞行时间装置，一个演示， 将进行结构分析。此外，各种 用于在线引入分离的肽级分的方法将 使用IT/reTOF的增强分辨率和速度进行研究。