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

这项拨款续期申请的目标是开发用于木质组学研究的新的质谱学技术。本文中的木质素组是给定样品中与木质素相关的化合物的全部，即所有木质素单体、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。它很难通过化学或生化方法降解。然而，解聚对于从木质素废料中回收有价值的化学品和生物燃料是极其重要的。在第一个资助阶段，我们开发了利用离子液体和高效催化活性电极进行木质素切割的新型电化学方法。质谱学(MS)分析表明，电化学降解产生了大量的击穿产物，通常是&gt；5000化合物，可以通过超高分辨率MS很容易地对其进行表征并将其归类为化学类别。我们开发了强大的二维可视化工具，提供了关于转化和确切的降解机理以及裂解产物的身份的详细信息。尽管有了强大的方法，但MS还不能表征完整的木质组，并且在分析非常低的丰度物种以及异构体和异构体方面也存在缺陷。在这项授权续期申请中，将开发新的鸟枪MS方法，这些方法将比目前可能的更深入地挖掘木质组，并产生关于木质素亚基的定量信息。特别是，由于木素的异质性、交联性和缺乏线性重复模式，目前从头测序非常困难，这将目前的技术限制在选择的小寡聚体上。我们正在计划通过新的串联-MS技术进行全面的实验，即所谓的“电子激活解离(EXD)”，并期待关于木质素亚基的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，我们的策略还包括离子迁移率光谱分离，在MS分析之前分离大量的异构体和异构体。最后，通过为复杂的质谱学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，避免现有的湿化学程序的问题，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.