Novel MS-based analytical approaches for shotgun lignomics and quantification of lignin

这项拨款更新申请的目标是为木质素组学研究开发新的质谱技术。在这种情况下，木质素组是给定样品中木质素相关化合物的完整补充；也就是说，所有的木质素单体、低聚物和聚合物。木质素是木本植物的主要成分，也是含量第二丰富的天然聚合物。它很难用化学或生化方法降解。然而，解聚对于从木质素废物中回收有价值的化学品和生物燃料非常重要。在第一个资助阶段，我们通过离子液体和高效催化活性电极开发了木质素裂解的新型电化学方法。质谱（MS）分析表明，电化学降解产生了大量的分解产物，通常有5000个化合物，可以很容易地通过超高分辨率质谱进行表征和划分化学类别。我们开发了强大的二维可视化工具，提供了详细的转化信息和精确的降解机制，以及解理产物的身份。尽管有这种强大的方法，但质谱还不能完全表征木素，并且在分析丰度非常低的物种以及同分异构体和等压线方面也存在不足。在这次拨款更新申请中，将开发新的霰弹枪质谱方法，比目前可能更深入地挖掘木质素组，并产生木质素亚基的定量信息。特别是从头开始的木质素测序目前非常困难，因为它的异质性，交联和缺乏线性重复模式，限制了目前的技术选择，小的低聚物。我们正计划通过新型串联质谱技术，即所谓的“电子激活解离（ExD）”，进行全面的实验，并期望获得有关木质素亚基不同连接的详细信息，从而增强木质素测序。为了获得更好的序列覆盖率，我们的策略还包括离子迁移率光谱分离，在质谱分析之前分离众多异构体和等压线。最后，通过为复杂的质谱原始数据开发增强的数据挖掘策略，我们将开发新的定量工具，避免现有湿化学程序的问题，并允许改进木质素亚基的定量测定。

The goal of this grant renewal application is the development of novel mass spectrometric techniques for lignomics research. The lignome in this context is the full complement of lignin-related compounds in a given sample; that is, all lignin monomers, oligomers and polymers. Lignin is a major component of woody plants and second most abundant natural polymer. It is difficult degrade by chemical or biochemical methods. Depolymerization, however, is extremely important for recovery of value chemicals and biofuels from lignin wastes. During the first funding phase, we have developed novel electrochemical methods for lignin cleavage by means of ionic liquids and efficient catalytically-active electrodes. Mass spectrometric (MS) analysis showed that electrochemical degradation yielded a very large number of breakdown products, typically >5000 compounds, which could be readily characterized and assigned to chemical classes by means of ultra-high resolution MS. We developed powerful two-dimensional visualization tools that provided detailed information on the transformation and exact degradation mechanisms as well as on the identity of the cleavage products. Despite the powerful approach, MS is not yet capable to characterize full lignomes and also exhibits shortcomings for analysis of very low abundant species as well as isomers and isobars. In this grant renewal application, novel shotgun MS methods will be developed that dig much deeper into the lignome than presently possible and also yield quantitative information on lignin subunits. In particular de novo lignin sequencing is currently very difficult because of the heterogenous nature, the crosslinking and the lack of linear repeat patterns, limiting the technique currently to selected, small oligomers. We are planning comprehensive experiments by means of novel tandem-MS techniques, so-called “electron-activated dissociations (ExD)”, and expect detailed information on the different linkages of lignin subunits and thus enhanced lignin sequencing. To obtain even better sequence coverage, our strategy also comprises ion mobility spectrometry separations, to separate the numerous isomers and isobars prior to MS analysis. Finally, by developing enhanced data mining strategies for the complex mass spectral raw data, we will develop new quantitative tools that avoid the problems of existing wet chemistry procedures and permit improved quantitative determination of lignin subunits.