Carbohydrate Polymers 251 (2021) 117080

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Structural elucidation of a branch-on-branch β-glucan from Hericium

erinaceus with A HPAEC-PAD-MS system
Bingying Xie a, 1, Lin Yi a, 1, Yiting Zhu b, Xiaoming Chang c, Jie Hao c, Li Pang c, Yilan Ouyang a,
Sheng Yuan b, Zhenqing Zhang a, *
a
Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou,
Jiangsu, 215021, China
b
Jiangsu Key Laboratory for Microbes and Microbial Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of Microbial
Resources, College of Life Science, Nanjing Normal University, Nanjing, 210023, China
c
Shanghai Green-Valley Pharmaceutical Co. Ltd., Shanghai, 201200, China

A R T I C L E I N F O A B S T R A C T

Keywords: A water-soluble β-glucan from fruiting body of Hericium erinaceus was obtained after water extraction, purifi­
Hericium erinaceus cation and fractionation. Analyses of monosaccharide composition, molecular weight and linkage mode
β-Glucan demonstrated that it is a β-glucan with 1→3 and 1→6 linkage modes and a molecular weight of 13.3 kDa. An
Sequence analysis
endo-1,6-β-D-glucanase was used to digest the β-glucan and the digested products over time were analyzed with a
HPACE-PAD
MS/MS
HPAEC-PAD-MS platform. The linkage mode of each glycosidic bond in the digested oligosaccharides were
confirmed with MS/MS. At the end of digestion, enzyme resistant oligosaccharides were observed as several 1→6
linked glucoses with one or two 1→3 linked glucose residues. All these domains are more like constructional
pieces from a branch-on-branch glucan, in which multiple 1→6 linked glucan chains are hooked through one or
two 1→3 linked glucose residues. Averagely, there is a 1→3 linkage per six 1→6 linked glucoses in this branch-
on-branch β-glucan.

1. Introduction
Hericium erinaceus (Bull.) Pers., also called Monkey’s mushroom,
Lion’s mane or Bear’s head in different places, belongs to class Basid­
iomycetes and family Hydnaceae (He et al., 2017; Lakshmanan et al.,
2016). It is mainly composed of fruiting body and mycelium as same as
other fungi in class Basidiomycetes (Ren, Perera, & Hemar, 2012). It is
usually regarded as a saprophyte growing on some types of dead timber
(Boddy, Crockatt, & Ainsworth, 2011). Actually, H. erinaceus was used
as both food and medicine as its excellent taste and nutritional function
during the past few centuries (Wang, Gao, Xu, Konishi, & Gao, 2014). It
was recorded in Compendium of Materia Medica that H. erinaceus could
aid digestion and nourish bodies. Its biological functions have also been
proved by modern pharmacology recently, such as H. erinaceus possesses
immunoregulation activity (Mizuno, Wasa, Ito, Suzuki, & Ukai, 1992;
Wu et al., 2018), hypolipidemic activity (Yang, Park, & Song, 2003),
hypoglycemic activity and neuroprotective activity (Cheng, Tsai, Lien,
Lee, & Sheu, 2016), as well as protection of gastrointestinal mucosa
(Khan, Tania, Liu, Rahman, & Mijanur, 2013; Shao et al., 2019; Wang
et al., 2018).
Among the bioactive components, polysaccharide in H. erinaceus has
been reported to play an important role in the activities mentioned
above, especially β-glucan (Khan et al., 2013). The β-glucan of
H. erinaceus was found to decrease blood sugar by increasing system
viscosity and coating starch, regulating glucose release and starch
digestion (Feng et al., 2019). In the case of potent immune-enhancing
effect, a β-glucan of H. erinaceus showed proliferation of lymphocytes
and up-regulation of inflammatory cytokines, such as IL-6, TNF-α and
IL-1β (Rodrigues et al., 2015; Wu et al., 2019). The β-glucans in fungi are
mainly derived from cell walls (Ruiz-Herrera & Ortiz-Castellanos,
2019). It is widely reported that most of those β-glucans have a large
diversity of molecular weight, α or β configurations, various types of
glycosidic bonds, different degrees of branching and so on (Synytsya &
Novak, 2013). Some studies showed the structure of β-glucan in
H. erinaceus as a (1→3, 1→6)-β-glucan (He et al., 2017; Ookushi, Saka­
moto, & Azuma, 2008; Ookushi, Sakamoto, & Azuma, 2009), and some

* Corresponding author.
E-mail address: z_zhang@suda.edu.cn (Z. Zhang).
1
These two authors contribute to this paper equally.

https://doi.org/10.1016/j.carbpol.2020.117080
Received 2 April 2020; Received in revised form 3 September 2020; Accepted 7 September 2020
Available online 16 September 2020
0144-8617/© 2020 Elsevier Ltd. All rights reserved.
B. Xie et al.
showed it was a 1→3-β-glucan with 1→6 β-glucopyranosyl
side-branches (Wu et al., 2019).
Better understanding of the structure of β-glucan is fundamental to
take a deep insight into its functions, but its complexity interferes with
the understanding its accurate structure. Nuclear magnetic resonance
(NMR) spectroscopy and methylation followed by gas chromatography –
mass spectrometry (GC–MS) are two methods widely used to elucidate
the structure of β-glucan from H. erinaceus (Wang, 2004; Wu, Zhou,
Zhou, Ou, & Huang, 2017; Zhang, Deng, Sun, Meng, & Zhang, 2011).
However, the requirement of sample amount and signals overlapping
limit the application of NMR for polysaccharide structure elucidation
(Duus, Gotfredsen, & Bock, 2000). Moreover, it mainly provides linkage
information but little sequence information (Matamoros Fernandez,
Obel, Scheller, & Roepstorff, 2004; Rong et al., 2017). Multiple harsh
and rigorous chemical reactions are involved in the process of methyl­
ation, which could lose some structural information and introduce po­
tential impurities (Kim, Reuhs, Michon, Kaiser, & Arumugham, 2006;
Sims, Carnachan, Bell, & Hinkley, 2018). Therefore, the followed
GC–MS analysis cannot provide accurate composition and linkage in­
formation of polysaccharides. The structure of β-glucan in H. erinaceus,
β-(1→6) and/or β-(1→3) linked glucan chain with diverse branches in
position O-3 or O-6, was widely reported with NMR and methylation
followed by GC–MS in previous studies (Geng, Liang, Zhao, & Zhang,
2004; Lee, Min, Cho, & Hong, 2009; Mori et al., 1998). However, the
results show scarcely any information about sequence or detailed
characterization of structural domains.
Bottom-up strategy is an important way to obtain sequencing infor­
mation of a polysaccharide (Solakyildirim, 2019; Zhang, Singh, &
Reinhold, 2005). Degradation and analytical method are two important
parts of this strategy. Enzyme is an ideal tool to degrade polysaccharide
comparing to chemical degradation, which has good specificity and
gentler reaction. The highly resolved separation technique plus mass
spectrometry is a good choice to analyze generated oligosaccharides
qualitatively and quantitatively. The structure and sequence of the
polysaccharide could be estimated according to the comprehensive an­
alyses and combination of the sequence information of each structure
domain.
In this work, a β-glucan was extracted, fractionated and purified from
H. erinaceus. Monosaccharide analysis and NMR were applied to confirm
its purity, composition and configuration. Prof. Yuan at Nanjing Normal
University provided an endo-1,6-β-D-glucanase kindly. This enzyme was
used to degrade the β-glucan of H. erinaceus to oligosaccharides specif­
ically. The products were systematically analyzed with high perfor­
mance anion exchange chromatography (HPAEC) with pulsed
amperometric detection (PAD), in parallel with electrospray ion quad­
rupole time-of-flight mass spectrometry (ESI-Q/TOF-MS). It is a
comprehensive analytical platform, which was developed and applied in
our lab for several studies (Rong et al., 2017; Yi, Ouyang et al., 2015). It
will provide the oligosaccharide mapping and the linkage information of
each separated oligosaccharide fragment. Based on these results, the
detailed structure of the β-glucan in H. erinaceus, including sequences of
various structural domains and the possible branch-on-branch structure,
will be characterized.
2. Materials and experiments
2.1. Materials
H. erinaceus was supplied by Jiancheng traditional Chinese medicine
pieces Co., Ltd. (Anhui, China). The endo-1,6-β-D-glucanase AfNeg1
from Aspergillus fumigatus (Boisramé & Gaillardin, 2009) was kindly
provided by Prof. Sheng Yuan from the Laboratory for Microbes and
Microbial Functional Genomics of Nanjing Normal University (Nanjing,
China). The enzyme was prepared accordingly and its specificity has
been tested by Prof. Yuan. It works on 1,6-β-glucan, but hard to digest
glucans with other linkage modes, like barley β-glucan (1→3 and 1→4
2
Carbohydrate Polymers 251 (2021) 117080
linked glucan), laminarin (1→3 linked glucan), etc (data not shown).
DEAE Sepharose fast flow was purchased from GE Healthcare Life Sci­
ence (Uppsala, Sweden). The monosaccharide (D-Gal, D-Glc, L-Fuc) and
3-(trimethylsilyl) propionic-2,2,3,3-d4 acid sodium (TSP-d4, 98 atom%
D) were purchased from Sigma-Aldrich (USA). Sodium hydroxide solu­
tion 50 % was purchased from Merck (Darmstadt, Germany).
High-purity water (resistivity≥18.2 MΩ×cm, 25 ◦ C) was used
throughout the study. All other chemicals and reagents were of HPLC
grade.
2.2. Extraction, purification and fractionation of β-glucan from
H. erinaceus
Slices of fruiting body of H. erinaceus were extracted twice with
distilled water at a ratio of 1:20 (w/v) at 100 ◦ C for 2 h. The combined
supernatant was concentrated to about one-tenth of the initial volume
with a rotary evaporator. The crude polysaccharide was precipitated
after 95 % ethanol was added. The crude polysaccharide was re-
dissolved with distilled water before proteins were digested with
papain. Remaining and/or digested proteins, peptides were removed
with Sevag reagent (Staub, 1965). The polysaccharides with higher
purity were precipitated with ethanol at 4 ◦ C overnight.
The purified polysaccharide was loaded onto a DEAE Sepharose Fast
Flow column (2.5 cm × 30 cm). The mobile phases were distilled water
and 1 M NaCl, respectively. Stepwise gradient (0, 5, 10, and 20 % of 1 M
NaCl) was employed to achieve four fractions. The fraction collector was
set as 10 mL/tube. The content of carbohydrate in each tube in fraction
collector was reflected by the responses of phenol–sulfuric acid assay.
The chromatogram was plotted with the colorimetric responses of each
tube as a function of elution time. The fractions were pooled, concen­
trated, desalted and lyophilized.
2.3. Monosaccharide composition analyses of β-glucan from H. erinaceus
The method used to analyze monosaccharide composition was
modified slightly based on the previously report (Zhang, Khan, Nunez,
Chess, & Szabo, 2012). Each sample was dissolved in 1 M trifluoroacetic
acid (TFA) to afford a solution with a concentration of 5 mg/mL and
then incubated at 100 ◦ C for 12 h. Excess TFA in each sample was
removed with a rotary evaporator under vacuum. The experiments were
performed on a Metrohm 850 Professional system equipped with a 919
IC auto-sampler plus, dual pumps and a PAD (Herisau, Switzerland).
Monosaccharides were separated on a CarboPac PA-1 column
(4.6 × 250 mm, Dionex, Thermo Fisher, USA) at a flow rate of 1 mL/min
with gradient elution, in which 100 % mobile phase A (15 mM NaOH)
was used in first 10 min and mobile phase B (150 mM NaOAc dissolved
in 15 mM NaOH) was increased from 0 to 35 % in following 10 min. The
data was acquired and analyzed with software MagIC Net 2.4 (Herisau,
Switzerland).
2.4. SEC-MALLS-RI analyses
The weight-average molecular weight of the water-soluble β-glucan
was determined by size exclusion chromatography with a multi-angle
laser light scattering and a refractive index detector (SEC-MALLS-RI)
as described in our previous work (Ouyang et al., 2017). All SEC was
performed on an Agilent 1260 system equipped with dual pumps (Agi­
lent, Santa Clara, CA). The separation was achieved on a size exclusion
column (Waters, Millford, MA, SEC BEH 200 Å, 1.7 μm, 4.6 × 150 mm)
at 0.1 mL/min with an isocratic mobile phase (50 mM NH4OAc). The
injection volume was 20 μL of each sample (10 mg/mL). The column
temperature was set at 30 ◦ C. The SEC system was coupled with a
MALLS (DAWN HELEOS II, Wyatt Technol., Goleta, CA) and a RI (Wyatt,
Optilab T-rEX) detector to analyze the MW. Data of the analysis was
manipulated by Wyatt ASTRA 6.1 software.
B. Xie et al.
2.5. NMR spectroscopy analyses
The polysaccharide sample (~70 mg) was dissolved in 0.8 mL D2O
and added 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid sodium as an
internal reference in a NMR tube. All experiments, including one-
dimensional spectra 1H, 13C NMR and two-dimensional spectra 1H-1H
correlated spectroscopy (1H-1H COSY), heteronuclear single-quantum
correlation (HSQC), were performed on a 600 MHz NMR spectrometer
(Bruker, Germany). 1H NMR spectrum was collected with 16 scans at
25 ◦ C and 65 ◦ C, while 13C NMR with 10240 scans and at 25 ◦ C. The
NMR data was processed with MestReNova software.
2.6. Digestion of the β-glucan by endo-1,6-β-D-glucanase
Approximately 40 mg β-glucan was dissolved in 5 mL 100 mM so­
dium acetate buffer (pH 5.0) and incubated with 0.35 U endo-1,6-β-D-
glucanase at 40 ◦ C. The aliquots were taken at 1, 6, 12 and 24 h. Another
0.35 U endo-1,6-β-D-glucanase was added in after 24 h, and the fifth and
sixth aliquots were taken at 36, 48 h. The aliquots were boiled at 100 ◦ C
for 20 min to inactivate enzyme and then centrifuged at 14000 rpm for
12 min to remove the denatured enzyme.
2.7. HPAEC-PAD-MS setting-up
The experiments were performed on the Metrohm 850 Professional
HPAEC system hyphenated an Agilent 6540 Q/Tof-MS system. Oligo­
saccharides in each aliquot were separated on a CarboPac PA-1 column
(4.6 × 250 mm, Dionex, Thermo Fisher, USA) at a flow rate of 1 mL/
min. Mobile phase A was 100 M NaOH and B was 1 M NaOAc dissolved
in 100 mM NaOH. The mobile phase B ascended from 0% to 15 % in
45 min.
A T-piece was connected behind the column to split the eluent and
about 70 % went to PAD, 30 % went to the desalter. The flow rate of each
channel depended on the length and aperture of tubes used in two
pathways. The desalter was essentially an ion suppressor MSM-HC Rotor
A (Metrohm, Switzerland), which was employed to remove sodium
before MS analysis. Once the eluent was desalted, it became water and
acetic acid. More details about the desalter were depicted in the previous
work of our laboratory (Yi, Ouyang et al., 2015).
MS data was collected in negative ion mode. Nitrogen gas was used
in the nebulizer at a pressure of 40 psi as drying gas. The flow rate of
nitrogen gas was 10 L/min at 350 ◦ C in the drying process and the
capillary voltage was set at 3.5 kV. The fragment voltage was set to 80 V.
A full MS scan between 100 and 2000 m/z was performed. The collision-
induced dissociation (CID) energies used in MS/MS to dissociate disac­
charide, trisaccharide, tetrasaccharide, pentasaccharide, hex­
asaccharide and heptasaccharide were set as 10− 45 V according to
different molecular mass. All data were acquired at negative ion mode
with Mass Hunter 6.0 (Agilent Technologies).
3. Results and discussion
3.1. Extraction, purification and fractionation of the β-glucan from
H. erinaceus
Crude polysaccharides were isolated from fruiting bodies of
H. erinaceus with a yield of 5% (w/w). After purification, four fractions
were eluted from DEAE Sepharose FF column with water (HEP1),
0.05 M (HEP2), 0.1 M (HEP3) and 0.2 M NaCl (HEP4), respectively. The
chromatogram is shown in Supplementary Fig. 1. Due to large dead
volume of the preparative column and tubes, the retention time of the
peaks lagged behind the time gradient changed. After dialysis and
lyophilization, the sugar content of each fraction was determined with
phenol–sulfuric acid assay using glucose as standard (Dubois, Gilles,
Hamilton, Rebers, & Smith, 1951). They were 105.5, 96.5, 92.0, and
87.8 % (w/w), respectively. The longer retention time and the lower
3
Carbohydrate Polymers 251 (2021) 117080
carbohydrate content in HEP4 implied that some charged components,
such as covalently linked protein or peptides were remained in HEP4. It
will not be considered any more in this work. In this study, we mainly
focused on HEP3 fraction.
3.2. Monosaccharide composition analyses of β-glucan from H. erinaceus
The chromatograms of monosaccharide compositions of four frac­
tions are shown in Supplementary Fig. 2. Both HEP1 and 2 were
composed of small amounts of fucose and galactose, as well as a big
amount of glucose, while HEP3 was composed of glucose exclusively
suggesting it was a homogenous glucan. Thus, the structure elucidation
was focused on the fraction HEP3 in this work.
3.3. Average molecular weight and molecular weight distribution of
β-glucan
The Mw of the β-glucan was analyzed as 13.3 kDa using SEC-MALLS-
RI. Only one normal distributed peak was observed in chromatogram of
HEP3, along with a low polydispersity index (Mw/Mn, 1.3) suggesting
the homogeneity of the β-glucan (Supplementary Fig. 3).
3.4. NMR analyses of HEP3
To determine the configuration and glycosidic linkage patterns of
this glucan, 1D and 2D NMR experiments were carried out. The spectra
of 1H and 13C NMR as well as 1H-1H COSY and 1H-13C HSQC at 25℃ are
shown in Fig. 1A–D. The 1H and 13C signals of HEP3 were assigned ac­
cording to their chemical shifts, relationships in 2D NMR spectra and the
previously reported works about other glucans (Kono et al., 2017;
Samuelsen et al., 2019). The assignments are listed in Table 1. Two
signals overlapped at around δ4.5 ppm were observed in anomeric areas
of 1H NMR spectrum (Fig. 1A). Besides, another cross peak with 1H
chemical shift at δ4.75 ppm emerged in HSQC spectrum (Fig. 1D), which
overlapped by HDO signal. All the proton signals mentioned above were
correlated with carbon signal at δ105.5 ppm, according to the cross
peaks in HSQC spectrum. Furthermore, chemical shifts of these
anomeric signals indicated a β configuration for all the glucopyranosyl
residues. Four sugar units were observed and labeled as a, b, c and d,
respectively. The signals, observed at δ4.55 and 4.51 ppm in Fig. 1A,
obtained the proton chemical shifts from COSY spectrum (Fig. 1C) as
3.53, 3.74, 3.51, 3.67, 4.22/3.82 ppm and 3.32, 3.48, 3.46, 3.62,
4.21/3.85 ppm for H2, H3, H4, H5, H6 and H6’, respectively. Then the
corresponding carbon chemical shifts could be clearly identified in
HSQC spectrum. As a result, the assignment of signals derived from
residue c and d completed successfully. Likewise, the assignment of
chemical shifts of residue a and b abided by the routine, as shown in
Table 1. The downfield shifting of carbon signal of position 3 (C-3)
suggested a substitution on hydroxyl group of position 3 of residues a
and c (Hu, Jiang, Huang, & Sun, 2016). The downfield shifting of C-6
suggested the substitution on position 6 of residues c and d (Liu et al.,
2018; Wu et al., 2004). All proton and carbon signals corresponding to
positions 2–6 observed at relatively higher magnetic field suggested no
other linkage on residue b, and they could be located at the terminals of
the glucan. The β configuration and 1→3/1→6 linkage patterns
observed in this glucan (HEP3) were as same as the properties of many
glucans from fungi and mushrooms reported previously (Bhunia et al.,
2010; Maity et al., 2011). Hence, it allowed assigning residues a, b, c and
d to →3)-β-Glc-(1→, β-Glc-(1→, →3,6)- β-Glc-(1→ and →6)-β-Glc-(1→,
respectively.
To solve the overlapping of HDO signal at δ4.8 ppm with the
anomeric proton signals at δ4.7− 4.8 ppm, another 1H NMR spectrum of
HEP3 was acquired at 65 ◦ C. The signal of HDO was shifted to higher
field and the anomeric proton signals were assigned and integrated
unambiguously. The ratio of the (1→3)-linked glucose and terminal
residues to the (1→6) and (1→3,6)-linked glucoses was 1:3. According
B. Xie et al. Carbohydrate Polymers 251 (2021) 117080

Fig. 1. NMR spectra of HEP3 and crude polysaccharide. (A) 1H-NMR spectrum of HEP3 at 25 ◦ C; (B) 13C-NMR spectrum of HEP3 at 25 ◦ C; (C) 1H-1H COSY spectrum
of HEP3; (D) 1H-13C HSQC spectrum of HEP3; (E) 1H-NMR spectrum of HEP3 at 65 ◦ C; (F) 13C-NMR spectrum of crude polysaccharide at 25 ◦ C.

to the linkage mode and each proportion, the β-glucan could be tenta­
tively deduced as a (1→6)-linked glucan main chain with (1→3)-linked
glucopyranosyl branching unit(s). Besides, a 13C NMR spectrum of the
crude polysaccharide is shown in Fig. 1F, which was similar to that of
HEP3, apart from some signals assigned to fucose and galactose e.g. δ74
ppm from HEP1 and 2. This reflects the proper fractionation.
3.5. HPAEC-PAD-MS analyses of digested products
The aliquots taken over digestion time were analyzed with the
HPAEC-PAD-MS system. Their PAD chromatograms are shown in Fig. 2.
The degree of polymerization (dp) of oligosaccharides corresponding to
each peak was confirmed with online MS analysis in negative mode.
Smaller oligosaccharides were eluted earlier, while the bigger ones had
longer retention time. Fair amount of mono- and disaccharides were

Table 1
1
H and 13C NMR chemical shifts of β-glucan from H. erinaceus in D2O at 25 ◦ C (δ, ppm).
Residue H1/C1 H2/C2 H3/C3 H4/C4 H5/C5 H6/C6

→3)-β-Glc-(1→ 4.78/105.5 3.53/75.8 3.75/87.2 3.59/71.0 3.54/78.5 3.91,3.72/63.6

β-Glc-(1→ 4.73/105.7 3.39/76.4 3.49/78.9 3.41/72.5 3.54/78.5 3.91,3.72/63.6
→3,6)-β-Glc-(1→ 4.55/105.8 3.53/76.0 3.74/88.2 3.51/71.2 3.67/77.5 4.22,3.82/71.8
→6)- β-Glc-(1→ 4.51/106.0 3.32/76.2 3.48/78.9 3.46/72.4 3.62/77.8 4.21,3.85/71.9

4
B. Xie et al.
Fig. 2. Overlaid HPAEC-PAD chromatograms of digested β-glucan products
taken from 1 h, 6 h, 12 h, 24 h and 36 h. (A) overall chromatograms recorded
from 0-47 min; (B) amplified chromatograms recorded from 13.5-47 min. dp,
degree of polymerization.
observed at 5 and 10 min, respectively, in the chromatogram of the 1 h
aliquot (Fig. 2A). Some regularly spaced oligosaccharide peaks with low
intensity were also observed in the magnified chromatogram (Fig. 2B),
which suggested that these oligosaccharides could be enzyme derived
1→6 linked oligosaccharides. The mono- and disaccharide increased in
the aliquot taken at 6 h, and some irregularly spaced oligosaccharide
peaks with significant amount were observed in its chromatogram
(Fig. 2A and B). The oligosaccharides corresponding to the regular
spaced peaks in the chromatograms decreased or disappeared except
mono- and disaccharide, while the irregularly spaced peaks increased
over digestion time (Fig. 2A and B), suggesting that the enzyme derived
1→6 linked oligosaccharides digested by enzyme subsequently after
they were released from β-glucan, and some enzyme resistant domains
were generated and accumulated over time. According to online MS
analysis, two trisaccharides (labeled as dp3a and b), four tetra­
saccharides (labeled as dp4a-d), six pentasaccharides (labeled as dp5a-
f), four hexasaccharides (labeled as dp6a-e) and three hepta­
saccharides (labeled as dp7a-c) were observed.
The oligosaccharide corresponding to each PAD chromatographic
peak was analyzed with MS and MS/MS. The singly charged molecular
ions [M− H]− of dp2-dp7 were observed at m/z 341, 503, 665, 827, 989
and 1151 in corresponding MS spectra. Adduct molecular ions con­
taining two water molecules [M+2H2O-H]− always appeared along with
[M− H]− as the aqueous media and observed at m/z 377, 539, 701, 863,
1025 and 1187, respectively, in corresponding MS spectra (Table 2).
3.6. MS/MS analyses of oligosaccharides in digested products
The water adduct molecular ions, [M+2H2O-H] − , were selected for
the following MS/MS analyses. Nomenclature of Domon and Costello
was applied to assign the fragment ions in their MS/MS spectra (Domon
& Costello, 1988). All the molecular ions and fragment ions were listed
5
Carbohydrate Polymers 251 (2021) 117080
in Table 2.
The MS/MS spectrum of disaccharide is shown in Fig. 3A. Besides the
typical glycosidic bond cleavage ions, B1 (m/z 161) and C1 (m/z 179),
three cross ring cleavage ions, 0,4A2 (m/z 221), 0,3A2 (m/z 251), 0,2A2
(m/z 281) were observed corresponding to the reducing end residue.
The cross ring cleavage pattern suggested no glycosylation occurred at
hydroxyl groups at position 2, 3 or 4 of the glucose residue, which
indicated the 1→6 linkage in the disaccharide. The glycosylation at 6
position would not stabilize the sugar ring. Thus, a series of cross ring
cleavage ions could be observed in MS/MS spectrum (Tang, Mechref, &
Novotny, 2005; Yi, Ouyang et al., 2015; Yi, Sun et al., 2015). All the
molecular ions and fragment ions were labeled both in the MS/MS
spectrum and on the structure. In the MS/MS spectrum of dp3a (Fig. 3B),
two series of cross ring cleavage ions 0,4A, 0,3A, 0,2A were observed
corresponding to the middle and reducing end residues as the diagnostic
fragment ions. It confirmed 1→6 linkages of these two residues in dp3a.
Hence, the sequence of dp3a was assigned as G1-6G1-6G. Different from
dp3a, the absence of cross ring cleavage ions from the middle residue of
dp3b suggested the glycosylation on the hydroxyl group at position 3,
which stabilized corresponding related bond on the sugar ring (An &
Lebrilla, 2011; Quemener, Ordaz-Ortiz, & Saulnier, 2006), and the
sequence of dp3b was assigned as G1-3G1-6 G (Fig. 3C).
In the MS/MS spectrum of dp4a (Fig. 4A), the observation of diag­
nostic cross ring cleavage ions, 0,4A, 0,3A, 0,2A, at the three residues
excluding non-reducing end residue suggested 1→6 linkages between all
residues and sequence of dp4a was identified as G1-6G1-6G1-6 G. The
absence of any cross ring cleavage ions on particular residue(s) sug­
gested corresponding 1→3 linkage(s) (Fig. 4B–D and Table 2). Accord­
ing to those, the other three tetrasaccharides, dp4b-d, were assigned as
G1-3G1-6G1-6 G, G1-6G1-3G1-6 G and G1-3G1-3G1-6 G, respectively.
Six pentasaccharides were observed in the enzyme derived oligo­
saccharides (Fig. 2). In the MS/MS spectra of dp5a, b, e and f (Fig. 5A, B,
E and F), the diagnostic cross ring cleavage ions of A2, A3, A3/A4 and A2/
A4 were absent, respectively. It suggested that the 1→3 linkage occured
at second, third, third/forth and second/forth residue(s) from non-
reducing end of those pentasaccharides, respectively. Their sequences
are G1-3G1-6G1-6G1-6 G, G1-6G1-3G1-6G1-6 G, G1-6G1-3G1-3G1-6 G
and G1-3G1-6G1-3G1-6 G. Dp5c was identified as a homogeneous 1→6
linked pentasaccharide according to its MS/MS spectrum in Fig. 5C, in
which diagnostic A cross ring cleavage ions were observed at every
residue. In the MS/MS spectrum of dp5d (Fig. 5D), the absence of
glycosidic bond cleavage ions, B3 (m/z 485) and C3 (m/z 503), suggested
a branch on the third residue from the non-reducing end. And the
absence of any diagnostic cross cleavage ions at the branched residue
suggested glycosylation occurs at position 3 on that sugar residue. In
addition, the presence of cross cleavage ions 0,3A2 suggested the 1→6
linkage between the first and the second residues from the non-reducing
end. Thus, the sequence of dp5d could be G1-6G1-6(G1-3)G1-6 G or G1-
6G1-3(G1-6)G1-6 G. The structures of these six pentasaccharides are
shown in Fig. 5 and their MS/MS assignments are listed in Table 2.
Four hexasaccharides were observed in the enzyme derived oligo­
saccharides (Fig. 2). Dp6a was only observed in the 1 h digested prod­
uct. It was disappeared in the following digestion. Dp6a was believed as
a homogeneous 1→6 linked hexasaccharide, which was released from
the β-glucan by enzyme at beginning, and subsequently digested to
smaller oligosaccharides. The absence of glycosidic bond cleavage ions,
B4 (m/z 647) and C4 (m/z 665), suggested a branch located at second
residue from reducing end of dp6b (Fig. 6A). In addition, the absence of
A3 cross ring cleavage ions suggested 1→3 linkage at the third residue
from non-reducing end of dp6b. Its sequence could be G1-6G1-3G1-3
(G1-6)G1-6 G or G1-6G1-3G1-6(G1-3)G1-6 G. Complying with the MS/
MS assignment principle for 1→3 and 1→6 linkages, dp6c and d were
B. Xie et al. Carbohydrate Polymers 251 (2021) 117080

Table 2
Assignments of molecular and fragment ions in MS/MS spectra of digested β-glucan oligosaccharides.
0,4 0,3 0,3 0,4 0,3 0,2 0,4 0,3
precursor B1 C1 A2 A2 A2 B2 C2 A3 A3 A3 B3 C3 A4 A4
ions (m/z)a

dp2 377 161 179 221 251 281 323

dp3a 539 161 179 221 251 281 323 341 383 413 443
dp3b 539 161 179 — — — 323 341 383 413 443
dp4a 701 161 179 221 251 281 323 341 383 413 443 — 503 545 575
dp4b 701 161 179 — — — 323 341 383 413 443 485 503 545 575
dp4c 701 161 179 221 251 281 323 341 — — — 485 503 545 575
dp4d 701 161 179 — — — 323 341 — — — — 503 545 575
dp5a 863 161 179 — — — 323 341 383 413 443 — 503 545 575
dp5b 863 161 179 221 251 281 323 341 — — — 485 503 545 —
dp5c 863 161 179 221 251 281 323 341 383 413 443 485 503 545 575
dp5d 863 161 179 — 251 — 323 341 — — — — — — —
dp5e 863 161 179 221 251 281 323 341 — — — 485 503 — —
dp5f 863 161 179 — — — 323 341 383 413 443 485 503 — —
dp6b 1025 161 179 221 251 281 323 341 — — — — 503 — —
dp6c 1025 161 179 — 251 — 323 341 — — — 485 503 — 575
dp6d 1025 161 179 — — — 323 341 383 413 443 485 503 545 575
dp7b 1187 161 179 — 251 — 323 341 383 413 443 485 503 545 575
dp7c 1187 161 179 221 251 281 323 341 — — — 485 503 545 575

aThe precursor ions of these oligosaccharides are [M+2H2O–H]− .

assigned as G1-6G1-3G1-6G1-3G1-6 G and G1-3G1-6G1-6G1-3G1-6 G,
respectively.
Three heptasaccharides were observed in the enzyme derived oli­
gosaccharides (Fig. 2). Dp7a was only observed in the 1 h digested
product. It was disappeared in the following digestion. Dp7a was
believed as a homogeneous 1→6 linked heptasaccharide, which was
released from the β-glucan by enzyme at beginning, and subsequently
digested to smaller oligosaccharides. The MS/MS spectra of other two
heptasaccharides are also shown in Fig. 6. Their sequences were
assigned as G1-6G1-6G1-6G1-6G1-3G1-6 G and G1-6G1-3G1-6G1-6G1-
3G1-6 G, accordingly.
4. Discussion and conclusion
In our previous works, HPAEC-PAD-MS was applied to analyze
branch patterns of dextran and sequence of linear β-glucan from high­
land barley qualitatively and quantitatively (Rong et al., 2017). Same in
this work, HPAEC separated β-glucan oligosaccharides with high reso­
lution, the hyphenated MS provided compositional and structural in­
formation, and PAD responded to reducing end of each oligosaccharide
proportionally. It is the first time to elucidate the structure, especially
sequences of β-glucan from H. erinaceus comprehensively.
The possible structure of the β-glucan is regardless the ratio of the
enzyme resistant domains. The solid “G” are enzyme resistant domains;
the “G” with dot outline represent enzyme digested domains.
Glucose, 1→6 linked disaccharide, three trisaccharides, four tetra­
saccharides, six pentasaccharides, four hexasaccharides and three hep­
tasaccharides were observed in the products digested over time.
Glucose, 1→6 linked disaccharide and enzyme resistant structural do­
mains containing 1→3 links were accumulated and observed in the final
digested product. Their sketches are shown in Fig. 7A. It is believed that
a big amount of glucose and 1→6 linked disaccharide were released
from the 1→6 linked main chain of β-glucan, considering the enzyme
specificity. All enzyme resistant fragments were shown as two or three
short 1→6 linked sugar chains hooked up by 1→3 linkages vertically. All
these fragments are more like constructional pieces from a branch-on-
branch glucan, in which there are multiple 1→6 linked glucan chains
and they hooked each other through one or two 1→3 linked glucose
residues. A possible schematic plot is shown in Fig. 7B. What’ more,
oligosaccharides with larger dps could be observed in the final enzymed
product, but the insufficient quality of MS/MS spectra of them and
limitation of chromatographic resolution hinder the sequence
confirmation.
The PAD peaks corresponding to these enzyme resistant structural
domains in the final digested product were integrated and their peak
areas are listed in Table 3. The peak areas reflected their molecular ra­
tios. The numbers of 1→3 and 1→6 linked Glc residue(s) in each
observed structural domain in Table 3 are confirmed according to their
sequences and enzyme specificity. Thus, the ratio of 1→3 and 1→6
linked Glc residues in the final digestion product was roughly calculated
as 1:6 using the equation at the bottom of the Table 3. The ratio of 1→3
and 1→6 linked Glc residues was also reflected by NMR analyses. This is
implied that the β-glucan is composed of 1→6 linked Glc residues and
small amount of 1→3 linked Glc residues, which briefly agrees with the
ratio of 1:6 between 1→3 and 1→6 linked Glc residues in this β-glucan
resulted from the HPAEC-PAD-MS analytical platform.
In this work, some detailed sequence information of β-glucan from
H. erinaceus was illuminated with the platform of HPAEC-PAD-MS,
comprehensively. Based on these sequence information, it is estimated
that the β-glucan from H. erinaceus, a branch-on-branch glucan, is
composed of multiple 1→6 linked glucan chains and they were hooked
through one or two 1→3 linked glucose(s). Averagely, there is a 1→3
linkage per about six 1→6 Glc residues in the structure.
Author’s contribution
The author contributions of this paper is listed as bellow:
Investigation_Ms. Bingying Xie took charge of most investigation
work.
Investigation_Ms. Lin Yi is an important investigator who is an expert
on HPAEC-MS plat form.
Investigation_Ms. Yiting Zhu is also an important investigator and
worked on the enzyme preparation.
Investigation_Mr. Xiaobing Chang, Jie Hao and Li Pang worked on
the sample preparation and NMR investigation.
Invesitgation_Dr. Yilan Ouyang manages the laboratory and helped
on oligosaccharide analysis.
Resource and validation _Prof. Sheng Yuan provided the enzyme and
validated the whole work.
Supervision_Prof Zhenqing Zhang is the supervisor of this work.

6
B. Xie et al. Carbohydrate Polymers 251 (2021) 117080

0,2 0,4 0,3 0,2 0,4 0,3 0,2 0,4 0,3 0,2
A4 B4 C4 A5 A5 A5 B5 C5 A6 A6 A6 C6 A7 A7 A7

605
605
605
605
605 647 665 707 737 767
605 647 665 707 737 767
605 — 665 707 737 767
— — 665 707 737 767
— — 665 707 737 767
— — 665 707 737 767
— — — — — — — 827 869 899 929
— 647 665 — — — — 827 869 899 929
— 647 665 — — — 809 827 869 899 929
605 647 665 707 737 — 809 827 — — — 989 1031 1061 1091
— 647 665 — 737 — 809 827 — — — 989 1031 1061 1091

Fig. 3. Structures and MS/MS spectra of disaccharide and trisaccharides. (A) structure and MS/MS spectrum of dp2; (B) structure and MS/MS spectrum of dp3a; (C)
structure and MS/MS spectrum of dp3b. A represented cross ring cleavage, B and C represented glycosidic bond cleavage.

7
B. Xie et al. Carbohydrate Polymers 251 (2021) 117080

Fig. 4. Structures and MS/MS spectra of tetrasaccharides. (A) structure and MS/MS spectrum of dp4a; (B) structure and MS/MS spectrum of dp4b; (C) structure and
MS/MS spectrum of dp4c; (D) structure and MS/MS spectrum of dp4d. A represented cross ring cleavage, B and C represented glycosidic bond cleavage.

8
B. Xie et al. Carbohydrate Polymers 251 (2021) 117080

Fig. 5. Structures and MS/MS spectra of pentasaccharides. (A) structure and MS/MS spectrum of dp5a; (B) structure and MS/MS spectrum of dp5b; (C) structure and
MS/MS spectrum of dp5c; (D) structure and MS/MS spectrum of dp5d, (E) structure and MS/MS spectrum of dp5e; (F) structure and MS/MS spectrum of dp5f. A
represented cross ring cleavage, B and C represented glycosidic bond cleavage.

9
B. Xie et al. Carbohydrate Polymers 251 (2021) 117080

Fig. 6. Structures and MS/MS spectra of hexasaccharides and heptasaccharides. (A) structure and MS/MS spectrum of dp6b; (B) structure and MS/MS spectrum of
dp6c; (C) structure and MS/MS spectrum of dp6d; (D) structure and MS/MS spectrum of dp7b; (E) structure and MS/MS spectrum of dp7c. A represented cross ring
cleavage, B and C represented glycosidic bond cleavage.

10
B. Xie et al. Carbohydrate Polymers 251 (2021) 117080

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