Lignin Structural Units Quantification By Multiple Reaction Monitoring And Its Application

Lignin is a complex natural polymer found in vascular plants,primarily resulting from the oxidative coupling of p-coumaryl alcohol,coniferyl alcohol,and sinapyl alcohol.The presence of lignin also has an important influence on the biorefinery process of lignocellulosic biomass feedstocks.Therefore,a better understanding of lignin’s composition and structure is essential for the efficient and effective utilization of lignin and plant materials.GC-MS is one of the most widely used methods for qualitative and quantitative lignin analysis.Moreover,the Multi-Reaction Monitoring（MRM）mode of GC-MS can enhance the signal/noise（S/N）ratios and accuracy of detecting unknown targets while allowing a shorter GC program for quantitative determination to avoid problems of co-eluted contaminants.The application of such an advanced analytical method,with higher sensitivity and accuracy,will be beneficial to broaden lignin research,which is of great significance for the study of lignin biosynthesis,as well as subsequent conversion and utilization of lignin.Elucidation and analysis of the lignins,including their compositions,contents,and structures,is one of the critical indexes in evaluating lignin material,which is also indispensable for studying the lignification process.In this study,a tailored high-throughput method of thioacidolysis was developed by incorporating MRM detection was reported,aiming at a higher sensitivity for accurately and sensitively analyzing lignin-derived monomers of a large number of biomass samples.Several representative lignocellulose samples,including gymnosperm,angiosperm,and poaceae were used to test this high-throughput method.The obtained results demonstrated that the ratios of lignin monomer compositions determined by the newly developed high-throughput method were consistent with traditional procedure despite the slightly higher monomer yields measured.In particular,a small amount of S monomers was detected from Polytrichum and Pogonatum proliferum by using the newly developed high-throughput procedure,which was beyond the detection limit of traditional procedures.Therefore,this newly developed protocol is expected to find various applications in biomass material component analysis and processing,plant breeding,plant evolution,plant genetic engineering,bioprocessing of lignocellulose for efficient biomass utilization.During the lignification of grass cell walls,ferulate acts like a nucleation site that can cross-couple with monolignols to form dimeric products.Based on this characteristic of lignin in the grass,ferulate-sinapyl alcohol cross-coupling reaction and products were investigated in detail to better understand the nature and scope of lignin-ferulate interactions.The structure of each product was delineated by GC-MS and NMR characterizations.It was found that sinapyl alcohol readily reacts with ethyl ferulate producing various cross-coupled dimers,as well as some homo-coupled products from ethyl ferulate or sinapyl alcohol.SA-（β-O-4）-FA（compound 3-8）was identified for the first time in a vitro free radical biomimetic system and the homo-coupled SA-（β-O-4）-SA（compound 3-3）from sinapyl alcohol was accurately quantified as less than 5%,while the half-open syringaresinol 3-5 was discovered when dimerization of SA was catalyzed by peroxidase in a biomimetic buffer system.Moreover,one new compound 3-18 formed by the cross-coupled linkage SA-（β-5）-FA（de-protonated）was identified by GC-MS and NMR.Overall,the understanding of the structural information by the ferulate-sinapyl alcohol cross-coupling will greatly improve our knowledge about the lignification of grass cell wall,and also provides important linkage information for the design of model compounds for structural characterization of lignin.Based on the structural analysis of lignin thioacidolysis dimers obtained by thioacidolysis/Raney-Ni desulfurization,eight lignin-derived dimers were obtained after a series of synthesis,includingβ-1,β-β,4-O-5,5-5 linkages.These synthesized compounds were used as the standard,while n-docodiane(C₂₂H₄₆)acted as the internal standard.Therefore,the response factors（RFs）of thioacidolylated dimer standards in TIC and MRM modes were determined by GC-MS.Those obtained response factors of eight lignin-derived dimeric compounds in the MRM mode were close to those in the TIC mode.Furthermore,the response factors of S-（β-1）-G（compound 4-4）in the TIC and MRM modes were 1.25 and 1.13,respectively,while the response factors of S-（β-1）-S（compound 4-8）were 1.62（TIC）and 1.45（MRM）.Using the synthesized standard compounds and the response factors measured,corresponding dimer structures of lignin in different biomass materials were quantitatively analyzed,including hardwood and grass.Finally,the highly sensitive qualitative and quantitative method,incorporating MRM detection of lignin-derived monomer and dimer,was used to investigate the dynamic changes of lignin components of two common plants（Arabidopsis Thaliana and Populus tomentosa）during the growth process.As the growing time prolonged,the three kinds of thioacidolysis monomers released from Arabidopsis plants showed an increasing trend,and the fifth week may be a particular growth stage with an obvious turning point.What’s more,the H/G ratio has shown a decreasing-increasing,and then decreasing tendency throughout the growth process of Arabidopsis.Besides,the lignin-derived dimeric products from Arabidopsis thaliana at different growth stages were also analyzed by MRM mode.It was found that the dynamic change trend of main dimeric products was more complex.S-（β-1）-S,S-（β-1）-S/OH,and S-（β-β）-S structure could hardly be detected in the first eight weeks of Arabidopsis seedlings.For populus tomentosa,the ratio of lignin monomers（H/G/S）in different growth periods showed a dynamic trend.In addition,compared with the results of 2D HSQC analysis of cell wall soluble components,the higher H/G and S/G values of thioacidolysis with MRM mode demonstrate its outstanding sensitivity advantage.Different from Arabidopsis Thaliana,most of the lignin-derived dimers can be detected in the whole growth cycle of Populus tomentosa,as early as the tissue culture seedling stage,including the S-（β-1）-G（38.5%）,S-（β-5）-G（8.6%）,G-（4-O-5）-G（42.6%）,G-（5-5）-G（10.2%）compounds.However,it is worth noting that the change of each lignin-derived dimer is not simple or absolute,to some extent,this study reals the growth rhythm of Populus tomentosa.To sum up,a tailored,fast,and sensitive thioacidolysis method incorporating a multi-reaction monitoring mode of GC-MS for lignin structural unit（monomers and dimers）quantification was successfully established by applying synthesized lignin model compounds.This study not only promoted the understanding of the grass cell wall lignification process and the changes of lignin structure in Arabidopsis thaliana and Populus tomentosa during the whole life cycle by using the newly developed method,but also providing new insights for the lignin biosynthesis pathway and the regulation of lignification process in the future.