Microsequencing Via Continuous Flow Matrix-Assisted Laser Desorption Ionization in an Ion Trap/Reflectron Time-of-Flight Device

这项工作的目标是开发基于新仪器的方法学，该仪器可用于检测和结构分析超低水平的生物重要性分子，即肽和蛋白质酶。特别是，这将涉及接口的毛细管分离方法的离子阱存储/反射飞行时间质谱仪检测器使用连续流MALDI方法。 在该方法中，色谱或分离产物将通过流动探针直接递送到离子阱中，其中MALDI以类似于连续流FAB实验的方式从直接来自探针尖端的连续流动液体进行。将使用离子阱存储/反射器飞行时间装置来分析所产生的离子，所述离子阱存储/反射器飞行时间装置可通过离子阱的长时间存储和冷却效应与反射器飞行时间装置组合来在液体MALDI过程中提供增强的灵敏度和解决方案。此外，将证明当与来自毛细管柱的d~液流连接时，在高压下操作阱的优点。 IT/reTOF将显示通过阱的MS/MS能力提供结构分析。 基本方法将应用于通常产生小于3000 Da的片段的酶促酶解酶。此外，该方法的能力也将用于在某些情况下可能产生高达8-10 kDa的产物的化学酶解酶。这种方法将直接比较在线毛细管分析使用的电喷雾源接口的IT/reTOF也正在开发中，我们的实验室。将评价CF MALDI在鉴别、灵敏度以及在干扰盐和缓冲液存在下的执行能力方面用于消化物分析的具体优势。

The goal of this work is to develop methodology based on new instrumentation which can be used for detection and structural analysis of ultralow levels of molecules of biological importance, i.e peptides and protein digests. In particular, this will involve the interface of capillary separation methods to an ion trap storage/reflectron time-of-flight mass spectrometer detector using a continuous flow MALDI method. In this method the chromatographic or separation products will be delivered via a flow probe directly into the ion trap where MALDI is performed from a continuously flowing Liquid directly from the tip of a probe in a manner analogous to continuous flow FAB experiments. The ions generated will be analyzed using an ion trap storage/reflectron time-of-flight device which can provide enhanced sensitivity and solution in the liquid MALDI process via the long teem storage and cooling effects of the ion trap in combination with a reflectron time-of-flight device. In addition, the advantages of trap operation at high pressure when interfaced to d~ liquid flow from capillary columns will be demonstrated. The IT/reTOF will be shown to provide structural analysis via the MS/MS capabilities of the trap. The basic methodology will be applied to enzymatic digests which generally yield fragments that are less than 3000 Da. In addition, the capabilities of the method will also evaluated for chemical digests which may yield products up to 8-10 kDa in some cases. This method will be directly compared to on-line capillary analysis using an electrospray source interfaced to the IT/reTOF also being developed in our lab. The specific advantages of the CF MALDI for digest analysis in terms of identification, sensitivity and for its ability to perform in the presence of interfering salts and buffers will be evaluated.