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，可用于检测和分析具有生物重要性的超低水平分子，即多肽和蛋白质消化。特别是，这将涉及毛细管分离方法与使用连续流动MALDI方法的离子陷阱存储/反射飞行时间质谱仪探测器的接口。在这种方法中，层析或分离产品将通过流动探头直接输送到离子陷阱中，在离子陷阱中，MALDI直接从探头尖端的连续流动液体以类似于连续流动FAB实验的方式进行。将使用离子陷阱存储/反射飞行时间装置对产生的离子进行分析，该装置可通过离子陷阱的长时间存储和冷却效果与反射飞行时间装置相结合，在液态MALDI过程中提供更高的灵敏度和解决方案。此外，还将展示与毛细管柱中的d~液体对接时高压捕集操作的优点。IT/reTOF将通过陷阱的MS/MS功能提供结构分析。基本方法将应用于酶消化，其产生的片段通常小于3000Da。此外，还将评估该方法的能力，用于化学消化，在某些情况下可能产生高达8-10 kDa的产品。这种方法将直接与在线毛细管分析进行比较，该在线毛细管分析使用的是连接到我们实验室正在开发的IT/reTOF的电喷雾源。将评估CF MALDI用于消化分析在鉴定、灵敏度以及在干扰盐和缓冲液存在的情况下执行的能力方面的具体优势。

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.