Applications of New Mass Spectral Techniques

电喷雾电离质谱法（ESI/MS）、大气压化学电离质谱法（APCI-MS）、串联质谱法（MS/MS）、基质辅助激光解吸电离（MALDI）质谱法、用于元素组成测定的精确质量测量以及毛细管电泳（CE）和质谱法的结合仍然是当前关注的技术。由于用于传统MALDI/MS的低分子量基质比样品多出100至1000倍，因此基质中的离子通常会模糊光谱的较低质量范围，并使小分子（分子量范围= 200-1000）的分析出现问题。我们发现，小分子的最佳整体常规基质是3,4-二羟基苯甲酸（DHB），尽管它并非完美或适用于所有化合物。MALDI/MS使用特殊的能量传递表面和直接激光解吸电离（LDI），没有基质的存在，都显示出克服基质限制和促进小分子快速分析的巨大希望。LDI质谱可以从各种各样的分子结构中获得，包括那些不容易吸收紫外线激光能量的化合物。初步研究表明，LDI/MS在表征小分子的能力方面与传统的MALDI/MS相当。非常规MALDI基质（如石墨）也在本研究中进行了研究。使用铅笔芯中的石墨获得的MALDI光谱显示出广泛的碱金属离子阳离子化，并且根据铅笔芯的来源显示出实质性的变化。然而，在表征小分子的能力方面，性能最好的石墨基体似乎与传统MALDI/MS具有可比性和互补性。采用盲对照化合物的更全面的评价正在进行中，目的是选择提供明确分子量信息的最佳机会的MALDI或LDI方法。此外，MALDI/MS与高效液相色谱（HPLC）的离线结合正在研究中，作为复杂生物和合成混合物靶向分析的替代方法。该方法包括在MALDI样品板上自动收集和定位HPLC洗脱液以及适当的基质，用于后续的质谱分析，包括精确的质量测量和源后衰减碎片分析或质谱/质谱分析。与在线LC/MS相比，这种方法的一个优点是生成原始分离样品的存档以供重新分析。合成衍生物建立在约束二酰基化甘油支架（dag -内酯）是有效的激动剂的蛋白激酶C （PK-C）。根据取代基R1和R2对应的酰基结构，这些dag -内酯表现出不同程度的PK-C同工酶特异性，并产生多种独特的生物活性。固相组合方法已在LMC中应用，以研究R1和R2的化学多样性，以产生更特异性的C1结构域配体。我们已经应用了多种质谱分析策略，在生物评价之前快速表征这些合成文库。单个dag -内酯的流动注射APCI/MS产生由MH+以及各种片段和溶剂加合离子组成的光谱。后一种方法，特别是如果使用MS/MS技术，在光谱信息方面似乎是FAB/MS的补充，尽管它不适合混合分析。然而，流动注射APCI/MS可实现自动化，并允许在无人值守的情况下对同一样品进行多次分析。MALDI/MS作为快速表征这些小分子文库的工具也很有用，特别是当使用铅笔芯石墨基体时（见上文）。作为一种快速和常规的分析筛选方法，基质优化和样品沉积过程的自动化正在研究中。我们的目标是在一天内对所有96个库组件进行全面的结构表征。

Electrospray ionization mass spectrometry (ESI/MS), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), tandem mass spectrometry (MS/MS), matrix-assisted laser desorption ionization (MALDI) mass spectrometry, accurate mass measurement for elemental composition determination, and combined capillary electrophoresis (CE) and mass spectrometry remain the techniques of current interest. Because the low-molecular-weight matrices used for conventional MALDI/MS are in 100- to 1000-fold excess to the sample, the ions from the matrix often obscure the lower mass range of the spectrum and make the analysis of small molecules (molecular weight range = 200-1000) problematic. We have found that the best overall conventional matrix for small molecules is 3,4-dihydroxybenzoic acid (DHB), although it is not perfect or applicable to every compound. MALDI/MS using special energy-transferring surfaces and direct laser-desorption ionization (LDI) without the presence of a matrix both show substantial promise for overcoming matrix limitations and facilitating the rapid analysis of small molecules . LDI mass spectra could be obtained from a wide variety of molecular structures including those compounds that would not be expected to readily absorb energy from a UV laser. Initial studies have shown that LDI/MS is comparable to conventional MALDI/MS in its ability to characterize small molecules. Unconventional MALDI matrices (e.g. graphite) have also been investigated in this study. MALDI spectra obtained using graphite from pencil lead exhibit extensive alkali metal ion cationization and show substantial variation depending on the source of the pencil lead. However, the best performing graphite matrix appears to be both comparable and complementary to conventional MALDI/MS in its ability to characterize small molecules. A more comprehensive evaluation employing blind control compounds is ongoing with the goal of choosing the MALDI or LDI methods that give the best chance of providing unambiguous molecular weight information. In addition, the off-line combination of MALDI/MS with high-performance liquid chromatography (HPLC) is under investigation as an alternate approach for the targeted analysis of complex biological and synthetic mixtures. This approach involves automatically collecting and spotting the HPLC eluent along with an appropriate matrix on a MALDI sample plate for subsequent MS analysis, which can include accurate mass measurement and post-source decay fragmentation analysis or MS/MS. An advantage of this approach over on-line LC/MS is the generation of an archive of the original separated sample for re-analysis. Synthetic derivatives built on a constrained diacylated glycerol scaffold (DAG-lactones) are potent agonists of protein kinase C (PK-C). Depending on the structure of the substituents corresponding to the acyl groups R1 and R2, these DAG-lactones exhibit varying degrees of PK-C isozyme specificity and produce a multitude of unique biological activities. A solid-phase combinatorial approach has been applied in the LMC to investigate chemical diversity at R1 and R2 in order to produce more specific C1 domain ligands. We have applied a variety of mass spectral analysis strategies to rapidly characterize these synthetic libraries before biological evaluation. Flow-injection APCI/MS of individual DAG-lactones produces spectra that consist of MH+ as well as various fragment and solvent-adduct ions. This later approach, especially if MS/MS techniques are used, appears to be complementary to FAB/MS in terms of spectral information, although it is not suitable for mixture analysis. However, flow-injection APCI/MS is amenable to automation and allows multiple analyses of the same sample to be carried out unattended. MALDI/MS also appears useful as a tool for the rapid characterization of these small molecule libraries, especially when a pencil lead graphite matrix is used (see above). Optimization of matrix and automation of the sample deposition process are under investigation as a rapid and routine analytical screening method. Our goal is the comprehensive structural characterization of all 96 library components in one day.