基于富勒烯衍生物的MALDI-MS反应性基质研究及其在生物活性小分子快速检测中的应用

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has attracted great attentions in small molecule (<1000 Da) analysis recently due to its high throughput, high sensitivity and low sample consumption. However, matrix signals in low m/z range and poor reproducibility of MALDI-MS signals hampered its application to the qualitative and quantitative analysis of small molecules. Fullerene chemistry is a field of organic chemistry devoted to the chemical properties of C60 related macromolecules. Up to now, the functionalization of C60 has become mature and directional; people can synthesize C60 derivatives as desired. Moreover, C60 itself can serve as MALDI-MS matrix. Thanks to the properties of C60, the current project aims to design and synthesize a series of C60 derivatives that can act as reactive matrices to react with small molecules and move the molecular weight of target compounds into the high m/z region. By applying a pair of C60 homolog labeling reagents to derive small molecules in real sample and standard solution respectively, the matrix effect in low m/z region could be circumvented, the MALDI-MS signal variations could be corrected and also no additional matrix is required owing to the great laser desorption/ionization property of C60 derivatives themselves. Thus, a fast, economic and reliable qualitative and quantitative analysis of bioactive small molecules could be developed. The proposed strategy based on C60 derivatives as reactive matrices of MALDI-MS is promising to be a reliable high-throughput analytical method of small molecules.

基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)近年来在小分子分析领域的应用逐渐受到重视，然而由于低分子量区域的背景干扰和检测重现性的限制，MALDI-MS对小分子的定性定量能力尚显不足。富勒烯化学是研究以C60为代表的化学分支，目前已可以按照实际需要高效且定向地合成一系列功能化的C60衍生物，同时C60自身也可作为MALDI-MS的基质。本项目拟通过合成一系列以C60为基本结构单元的标记试剂，作为生物活性小分子的MALDI-MS“反应性基质”和质量位移试剂，并提供廉价易得的内标，从而解决MALDI-MS在小分子分析中存在的背景干扰和重现性差的问题，最终建立疾病相关的生物活性小分子准确、快速、经济的C60标记-MALDI-MS定量分析方法。本项目发展的以C60标记试剂作为MALDI-MS“反应性基质”的策略，有望使MALDI-MS成为小分子分析的高通量检测手段。

基质辅助激光解吸电离质谱具有样品消耗量少、高通量、高灵敏的优点，在多肽、聚合物等大分子分析领域应用广泛。然而，其在小分子分析领域的应用受到基质信号干扰严重及重现性不足的限制。为解决上述问题，本项目进行了以下研究：1)设计合成了琥珀酰亚胺-富勒烯衍生物作为氨基酸的质量位移试剂，氨基酸的标记产物质荷比位移至高质荷比区域，解决了传统有机基质带来的信号干扰问题；合成COOH-C60作为内标，有效地校正了质谱信号波动；通过引入上述C60标记试剂，成功建立了微升级体液中氨基酸的MALDI MS快速测定方法。2）设计合成了溴乙酰-富勒烯衍生物作为活性巯基小分子的质量位移试剂，建立了血清中活性小分子巯基化合物的MALDI MS快速分析方法；3)提出黑磷辅助激光解吸电离质谱法，该方法对季铵盐类小分子化合物表现出高选择性及高灵敏度，我们成功建立了唾液中醛酮化合物及血液中葡萄糖的MALDI MS快速高灵敏度测定方法；4）引入主体分子葫芦脲作为新型质量位移试剂，通过与化学衍生法结合，实现了多种小分子化合物的全面分析，建立了微量织物组织中多胺、唾液和中药中醛酮化合物的MALDI MS快速测定方法。本项目从引入新型质量位移试剂及小分子的新型无机基质两方面出发，为复杂基质小分子的MALDI MS分析研究提供了新的研究策略，研究成果有望用于临床分析等领域。