Department of Chemistry, Michigan State University, East Lansing, MI, USA.
Analyst. 2013 Nov 7;138(21):6683-92. doi: 10.1039/c3an36709f.
Recalcitrance of grasses to enzymatic digestion arises to a significant degree from a complex array of phenolic crosslinks between cell wall polysaccharide chains that inhibit their conversion to biofuels and lower their nutritive value for animal feed applications. Polysaccharide esters of ferulic acid are abundant in plant cell walls. Crosslinks between polysaccharides are formed through oxidative dehydrodimerization of ferulates, producing dehydrodiferulates (henceforth termed diferulates). Such ferulates and diferulates further crosslink plant cell walls by radical coupling cross-reactions during lignification. Although cell wall digestibility can be improved by cell wall metabolic engineering, or post-harvest by various pretreatment processes, a more comprehensive understanding of the role and impact of ferulate crosslinking on polysaccharide hydrolysis would be accelerated by availability of analytical methods that can distinguish the various diferulates released during biomass pretreatments, many of which are isomers. In this report, we present an ultrahigh-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) strategy for comprehensive separation and identification of diferulate isomers. Collision-induced dissociation (CID) mass spectra of M + H ions distinguished various isomers without requiring derivatization. Characteristic product ions for 8-O-4-, 8-8-non-cyclic, 8-8-cyclic, 8-5-cyclic, 8-5-non-cyclic, and 5-5-linked isomers were identified. All diferulates were identified either as di-acids in extracts of NaOH-hydrolyzed corn stover, or as a diverse group of diferulate mono- and di-amides in extracts of Ammonia Fiber Expansion (AFEX™)-treated corn stover. This approach allows for direct analysis of released diferulates with minimal sample preparation, and can serve as the foundation for high-throughput profiling and correlating pretreatment conditions with biomass digestibility in biorefineries producing biofuels and biochemicals.
草类对酶解的抗性在很大程度上源于细胞壁多糖链之间复杂的酚类交联，这些交联抑制了它们转化为生物燃料的能力，并降低了其作为动物饲料应用的营养价值。阿魏酸的多糖酯在植物细胞壁中含量丰富。多糖之间的交联是通过阿魏酸的氧化脱氢二聚化形成的，产生去氢二阿魏酸（以下简称二阿魏酸）。这些阿魏酸和二阿魏酸在木质化过程中通过自由基偶联交叉反应进一步交联植物细胞壁。虽然可以通过细胞壁代谢工程或收获后的各种预处理过程来提高细胞壁的可消化性，但如果能够获得可区分生物质预处理过程中释放的各种二阿魏酸的分析方法，将加速对阿魏酸交联对多糖水解作用和影响的更全面了解，其中许多二阿魏酸是同分异构体。在本报告中，我们提出了一种超高效液相色谱/串联质谱（UHPLC/MS/MS）策略，用于全面分离和鉴定二阿魏酸的同分异构体。M + H离子的碰撞诱导解离（CID）质谱可以区分各种同分异构体，而无需进行衍生化。鉴定出 8-O-4-、8-8-非环、8-8-环、8-5-环、8-5-非环和 5-5-连接的同分异构体的特征产物离子。所有的二阿魏酸都被鉴定为在氢氧化钠水解玉米秸秆提取物中的二羧酸，或者在氨纤维膨胀（AFEX™）处理玉米秸秆提取物中的各种二阿魏酸单酰胺和二酰胺。这种方法可以在最小的样品制备下直接分析释放的二阿魏酸，并且可以作为高通量分析的基础，将预处理条件与生物炼制厂生产生物燃料和生物化学物质的生物质消化率相关联。
