Applications of New Mass Spectral Techniques

该项目的目标是开发新的质谱学技术，以便为涉及(1)化学结构确定、(2)复杂混合物分析和(3)生物系统中痕量组分测量的问题提供创新和/或更快速的解决方案。基质辅助激光解吸电离(MALDI)质谱仪、电喷雾电离质谱仪(ESI/MS)、串联质谱仪(MS/MS)、液质联用(LC/MS)、毛细管电泳质谱(CE/MS)联用和精确质量测定是目前研究的热点。采用后续峰采集的在线CE样品浓缩技术正在研究中，作为CE与高分辨率MALDI/MS离线结合和源后衰变碎片分析的一种制备规模方法。还在研究将高效液相色谱与MALDI/MS的离线结合用于复杂合成混合物的分析。 一些建立在受限制的甘油支架(二取代DAG-内酯)上的合成化合物已被鉴定为蛋白激酶C(PK-C)的有效激动剂。根据包括R1和R2的取代基的结构，这些DAG-内酯似乎具有一定程度的PK-C同工酶专一性。一种固相组合方法正被应用于LMC中，以研究R1和R2的化学多样性，以产生更具体的C1域配体。重要的是，在生物评价之前，必须快速表征这些合成文库，并建立它们的结构。快速原子轰击质谱仪(FAB/MS)、流动注射ESI/MS、流动注射APCI/MS和MALDI/MS是对合成的DAG-内酯文库进行初步表征的快速质谱学方法。这些文库的快速、定性结构表征的一种策略是使用FAB/MS混合分析。由于典型的DAG-内酯衍生物的FAB质谱图既是可预测的，又是结构信息丰富的，它可以用来鉴定存在于简单混合物中的DAG-内酯衍生物。最初，选定的文库组分被单独检查，以评估化学效率并确认质谱图的行为与预测的一致。然后组装、分析和解释包含整个化学空间的简单的4到6组分的混合物。随后对混合物分析过程中未检测到的库组件进行单独分析。混合分析直接与单个分析相关，大大减少了要检查的样品数量，导致了更好的分析周转，并可进行准确的质量测量。流动注射电喷雾电喷雾质谱(ESI/MS)不适用于这些DAG-内酯类化合物的分析，因为它们的亲油性较高，且碱性有限。流动注射-APCI/MS产生由MH以及各种碎片和溶剂加成离子组成的光谱。在光谱信息方面，后一种方法似乎是对FAB/MS的补充。MALDI/MS似乎也是这些小分子文库快速表征的有用工具，尽管可变的碱金属离子阳离子作用使光谱解释变得复杂。目前正在研究无矩阵MALDI，作为解决这一问题的一种方法，并提供一种更快速的分析方法。我们的最终目标是在一天内完成所有96个库组件的结构特征。每个文库组件的等量都被存档，以备将来可能的分析，如果以后的生物学评估需要额外的特征。 FAB/MS通常用于通过对新化合物和合成中间体的结构表征来支持LMC的合成工作。

The objective of this project is development of new mass spectral techniques in order to provide innovative and/or more rapid solutions to problems involving (1) chemical structure determination, (2) complex mixture analysis and (3) measurement of trace components in biological systems. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry, electrospray ionization mass spectrometry (ESI/MS), tandem mass spectrometry (MS/MS), combined liquid chromatography-mass spectrometry (LC/MS), combined capillary electrophoresis-mass spectrometry (CE/MS) and accurate mass measurement are the techniques of current interest. On-line CE sample concentration techniques with subsequent peak collection are being investigated as a preparative-scale method for the off-line combination of CE with high resolution MALDI/MS and post-source decay fragmentation analysis. The off-line combination of high-performance liquid chromatography (HPLC) with MALDI/MS is also being investigated for the analysis of complex synthetic mixtures. A number of synthetic compounds built on a constrained glycerol scaffold (disubstituted DAG-lactone) have been identified as potent agonists of protein kinase C (PK-C). Depending on the structure of the substituents comprising R1 and R2, these DAG-lactones appear to have some degree of PK-C isozyme specificity. A solid-phase combinatorial approach is being applied in the LMC to investigate chemical diversity at R1 and R2 in order to produce more specific C1 domain ligands. It is important that these synthetic libraries be rapidly characterized and their structures established before biological evaluation. Fast atom bombardment mass spectrometry (FAB/MS), flow-injection ESI/MS, flow-injection APCI/MS and MALDI/MS have been investigated as rapid mass spectral approaches for the initial characterization of these synthetic DAG-lactone libraries. One strategy for the rapid, qualitative structural characterization of these libraries employs FAB/MS mixture analysis. Because the FAB mass spectrum of a typical DAG-lactone derivative is both predictable and structurally informative, it can be used to identify the DAG-lactone derivatives present in simple mixtures. Initially, selected library components are examined individually to evaluate the efficiency of the chemistry and to confirm that mass spectra behave as predicted. Simple 4- to 6-component mixtures encompassing the entire chemical space are then assembled, analyzed and interpreted. Library components not detected during mixture analysis are subsequently analyzed individually. Mixture analysis correlates directly with individual analysis, substantially reduces the number of samples to be examined, results in enhanced analytical turn-around and is amenable to accurate mass measurement. Flow-injection ESI/MS is not suitable for analysis of these DAG-lactones because of their high lipophilicity and limited basicity. Flow injection-APCI/MS produces spectra that consist of MH+ as well as various fragment and solvent-adduct ions. This later approach appears to be complementary to FAB/MS in terms of spectral information. MALDI/MS also appears useful as a tool for the rapid characterization of these small molecule libraries although variable alkali metal ion cationization complicates spectral interpretation. Matrix-less MALDI is currently under investigation as an approach to resolve this problem and provide an even more rapid analysis approach. Our ultimate goal is the structural characterization of all 96 library components in a one day. Aliquots of each library component are being archived for possible future analysis should later biological evaluation warrant additional characterization. FAB/MS is routinely employed to support the LMC synthetic effort through structural characterization of new compounds and synthetic intermediates.