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

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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.