METHODS DEVELOPMENT FOR SEQUENCING USING AN ION/TRAP

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 对肽和翻译后修饰肽进行测序 低皮摩尔水平及以下。此项工作将专门开展 用于对使用 2-D 凝胶分离的肿瘤相关蛋白进行测序 密歇根大学二维凝胶实验室的电泳。 这些蛋白质被酶促裂解成肽片段， 由此产生的肽通常不能轻易地通过 Edman 进行测序 由于 N 末端堵塞、样品量低或 无法检测翻译后修饰。 该提案将 因此涉及离子阱/反射时间的进一步开发 飞行质谱仪及各种能力的开发 这是这种混合仪器组合的结果。 这个装置 将用于获取肽消化物的序列信息 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。 离子阱将充当前端存储 用于飞行时间质谱仪的装置，具有长时间的能力 长期储存，从而提高检测的灵敏度 从 2-D 凝胶中获得超低水平的肽。 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 功能。长期 陷阱中大离子的存储将被证明是特别重要的 对于检测长寿命单分子碎片很重要 这些大型物种已被证明是在时间尺度上发生的 超出了大多数质谱仪的能力。 各种因素 允许足够的碎片来解释结构 将研究该过程中的肽。 此外， IT/reTOF 的捕获能力将提供足够的分辨率 解析肽分子离子的同位素分布 和它们的碎片峰，以便从 电喷雾电离可以根据其电荷来识别 州。 最终，通过使用观察到的碎片模式 反射器飞行时间装置，展示了以下能力 将提出获得结构分析。此外，各种 在线引入分离的肽级分的方法将是 使用 IT/reTOF 的增强分辨率和速度进行研究。

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.