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Mass Spectrometry-based sequencing of lignin oligomers
in bioenergy cropsKris Morreel*, Geert Goeminne*, Oana Dima*, Chiarina Darrah*, John Ralph** and Wout Boerjan*
* VIB - Department of Plant Systems Biology, Ghent University - 9052 Ghent, Belgium
** Department of Biochemistry and DOE Great Lakes Bioenergy Research Center, University of Wiconsin, Madison, Wisconsin 53706, USA
Phenolic profiling of bioenergy plant extracts by UPLC-MS
Introduction
Identification of oligolignols
containing alternative units
Conclusion
O
O
OH
OH
OMe
OMe
OMe
OH
OH
OH
O
OMe
OH
OH
OMeOH
OMe
OH
8 8
8 O 4
8 5
4
5
OMe
8
OH
OH
OMe
OH
OH
MeO
8
OH
OH
8-8
8-O-4
8-5
H G S
Monolignol free radicals undergo radical coupling reactions which
lead to a variety of dimers (dilignols). Dimerization can occur through
8–O–4, 8–5 or 8–8 coupling. These dilignols undergo further endwise polymerization
to form tri-, tetra-, ... oligolignols, which lead to the lignin polymer.
p-coumaryl alcohol Coniferyl alcohol sinapyl alcohol
Brachypodium (stem)Poplar (stem)
Sugarcane (stem)
OH
O
OMe
C
OH
O
OMeC
OH
O
OMe
CH
OH
O
OMe
CH
OH
O
OMe
MS/MS
G(8–O–4)G(8–O–4)G(8–O–4)G(8–O–4)G
MS/MS
Sequencing approach illustrated on the pentamer G(8–O–4)G(8–O–4)G(8–O–4)G(8–O–4)G. The number of
linkages can be deduced from the number of ion clusters. A, Sequencing starting from the 8-end using the B ion
series (yellow). First product ions due to pathway I and II fragmentations are shown (dark and light yellow). B,
Annotation of first product ions (dark and light purple, A ion series) after sequencing from the 4-end.
B
A
Based on the pathway I peak pattern
for β-aryl ethers, 134 oligolignols, from
trimers up to hexamers, were detected.
25 of the 134 oligolignols could be
completely sequenced by our approach
taking only G and S units into account.
Of the remainder, a complete sequence
was obtained for nine oligolignols when
taking the presence of a G′, S′, or SP
unit into account, and two structures
were resolved after taking a hexose or
methyl group into account. Still, 98
remained only partially sequenced. In
11 cases, this arose from the inability to
determine the correct position of some
of the units. Following a more elaborate
MSn analysis, two of these 11
oligolignols were shown to contain a
putative reduced phenylcoumaran or
resinol linkage unit. Preliminary
sequence terminations became more
frequent as sequences were longer (18
cases), requiring that MSn of particular
first product ions had to be performed.
However, the majority of the cases (69
cases) could not be sequenced due to
the presence of as yet unknown units
(annotated with an X).
Sequencing of oligolignols
Lignin is a major component of plant secondary cell walls, enabling water and nutrient transport
through the vascular system. Lignin is made from the combinatorial radical-radical coupling of
monolignols and many other less abundant monomers. This polymerization process leads to a
plethora of units and linkage types that affect the physicochemical characteristics of the cell wall.
Current methods to analyze the lignin structure focus only on the frequency of the major monomeric
units and interunit linkage types. We have now developed a mass-spectrometry-based tool to
determine the sequence of units in small lignin polymers as well as the bonds connecting these
units. This technique is now used to identify lignin polymers composed of as yet unknown units and
bond types. This knowledge will be instrumental in engineering plants with new lignin structures that
are more easily degraded during the pretreatment of plant biomass.
Lignin polymerization starts with radical-radical cross-coupling between three main end products of the phenylpropanoid pathway, p-coumaryl alcohol (H), coniferyl alcohol (G) and sinapyl alcohol (S). These interactions lead to the formation of small oligomers that represent the initial stages of lignin polymerization
Oligolignol isolation Oligolignol isolationOligolignol isolation
MeOH extracts of plant material from bioenergy plants contain hundreds of phenolic compounds, creating a very complex source of data for a targeted oligolignol analysis. Here we show the base peak ion chromatograms of MeOH extracts of bioenergy crops such as sugarcane, poplar and
Brachypodium in black. In green, the extracted chromatograms for theoretical oligolignols are presented. This theoretical list takes into account all possible combinations following 8–O–4, 8–5 and/or 8–8 coupling reactions between H, G and S units. The respective m/z values are calculated and
used for isolating all possible oligolignols from the raw data. These results reflect the wide variety of oligolignol types in different bioenergy crops. Identification of these oligolignols is done by sequencing through LC-MS/MS.
O
O
OH
OMe
MeO
OH
OH OH
OH
OH
OH
OMe
MeO
OH
O
OH
OH
OMeMeO
OH
O
OHOH
O
OH
O
OH
OH
OMeMeO
OMe
OH
Using high-resolution MS and MSn spectra, the structure of a more frequently encountered trimeric moiety in the total list
of oligolignols was unraveled: G(8–O–4)S(8–5)Gglycerol. Arylglycerol end units such as Gglycerol are known to occur as
minor structures in lignin .
Extracted ion chromatogram for m/z 963
Extracted ion chromatogram
for m/z 617.17
references Mass spectrometry-based fragmentation as an identification tool in lignomics Morreel K, Kim H, Lu F, Dima O, Akiyama T, Vanholme R, Niculaes C, Goeminne G, Inzé D, Messens E, Ralph J, Boerjan W .
ANALYTICAL CHEMISTRY, 2010, session ‘Breakthrough Technologies’
Mass spectrometry-based sequencing of lignin oligomers Morreel K, Dima O, Kim H, Lu F, Niculaes C, Vanholme R, Dauwe R, Goeminne G, Inzé D, Messens E, Ralph J, Boerjan W. PLANT PHYSIOLOGY, 2010
We have developed a method that allows, for the first time, to identify the sequence of lignin monomers and the bonds connecting them, in small lignin polymers. This breakthrough
technique will allow us to determine the structures of novel lignin polymers engineered to contain alternative monomer substitutes that create easy cleavable chemical bonds.
Through this technique we already unleashed 134 β-arylether- containing oligolignols in polar xylem extracts, the majority of which possessed and unknown unit and/or linkage structure.
OH
O
OH
I
II8
4
Lignin polymer