Applications of New Mass Spectral Techniques

该项目的目标是开发新的质谱技术，以提供创新和/或更快速的解决方案，解决涉及（1）化学结构测定，（2）复杂混合物分析和（3）生物系统中痕量成分的测量。基质辅助激光解吸电离（MALDI）质谱、电喷雾电离质谱（ESI/MS）、串联质谱（MS/MS）、液相色谱-质谱联用（LC/MS）、毛细管电泳-质谱联用（CE/MS）和精确质量测量是当前感兴趣的技术。在线CE样品浓缩技术与随后的峰收集正在研究作为一种用于离线组合CE与高分辨率MALDI/MS和源后衰变裂解分析的分析尺度方法。离线组合的高效液相色谱（HPLC）与MALDI/MS也正在研究复杂的合成混合物的分析。 许多建立在受限甘油支架上的合成化合物（二取代的DAG-内酯）已被鉴定为蛋白激酶C（PK-C）的有效激动剂。根据包含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同工酶特异性。一个固相组合的方法被应用在LMC研究化学多样性在R1和R2，以产生更具体的C1结构域配体。重要的是，这些合成库的快速特性，并建立其结构之前的生物学评价。快速原子轰击质谱（FAB/MS），流动注射ESI/MS，流动注射APCI/MS和MALDI/MS已被研究作为快速质谱方法的初步表征这些合成的DAG-内酯库。这些文库的快速、定性结构表征的一种策略采用FAB/MS混合物分析。因为典型的DAG-内酯衍生物的FAB质谱是可预测的和结构信息的，所以它可用于鉴定简单混合物中存在的DAG-内酯衍生物。最初，单独检查选定的文库组分以评估化学效率并确认质谱表现与预测一致。简单的4- 6组分混合物涵盖整个化学空间，然后组装，分析和解释。随后单独分析在混合物分析期间未检测到的文库组分。混合物分析与单独分析直接相关，大大减少了待检测样品的数量，提高了分析周转率，并可进行准确的质量测量。流动注射ESI/MS不适合分析这些DAG-内酯，因为它们的高亲油性和有限的碱性。流动注射-APCI/MS产生由MH+以及各种碎片和溶剂加合物离子组成的光谱。这后一种方法似乎是补充FAB/MS的光谱信息。MALDI/MS也似乎是一种有用的工具，这些小分子库的快速表征，虽然可变的碱金属离子阳离子化复杂的光谱解释。无矩阵MALDI目前正在研究中，作为解决这个问题的方法，并提供一个更快速的分析方法。我们的最终目标是在一天内对所有96个文库组分进行结构表征。将每个文库组分的等分试样存档，以备将来可能进行的分析，如果后续生物学评价需要进行额外表征。 FAB/MS通常用于通过新化合物和合成中间体的结构表征来支持LMC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.