Electrophoresis 2020, 0, 1–6 1

Bence Mátyás1,2 Research Article

Júlia Singer3
Máté Szarka4,5,6
Daniel A. Lowy1,2,7 Determination of complex type free,
Boglárka Döncző5
Péter Makleit8
non-conjugated oligosaccharide glucose
Virgilio E. Failoc-Rojas9
Andrés Ramirez10
unit values in tomato xylem sap for early
Pedro Martínez10 detection of nutrient deficiency
Zsolt Sándor2,8
Ida Kincses8
Although knowledge on glycan biosynthesis and processing is continuously maturing,
András Guttman4,11
there are still a limited number of studies that examine biological functions of N-glycan
1 Genesis Sustainable Future Ltd., structures in plants, which remain virtually unknown. Here, the statistical correlation be-
33 Rákóczi St., Sárospatak, tween nutrient (nitrogen) deficiency symptoms of crops and changes in 8-aminopyrene-
B-A-Z, H-3950, Hungary 1,3,6-trisulfonic acid (APTS)-labeled complex type free oligosaccharides is reported. While
2 Research Group of Applied
Plant Glycobiology, Dama deficiency symptoms are predicted by multispectral images and Kjeldahl digestion, APTS-
Research Center limited, labeled complex type free oligosaccharides are identified by their glucose unit (GU) values
Kowloon, Hong Kong in tomato xylem sap, using capillary electrophoresis with laser induced fluorescence de-
3 Accelsiors Ltd., Budapest,
tection (CE-LIF). Given the limited number of structures obtained from plants, archived
Hungary
4 Horváth Csaba Memorial in the literature, in the future, it is intended to create an open access database of promis-
Laboratory of Bioseparation ing indicators, namely, glycan structures that are presumably responsible for the nutrient
Sciences, Research Center for deficiency caused stress in plants (http://glycoplants.org).
Molecular Medicine, Faculty of
Medicine, Doctoral School of
Molecular Medicine, University Keywords:
of Debrecen, Hungary Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) /
5 Institute for Nuclear Research
Free oligosaccharides / N-glycosylation in plants / PCT-FNGs
(Atomki), Debrecen, Hungary DOI 10.1002/elps.202000254
6 Vitrolink Ltd., Debrecen,


Hungary
7 Northern Virginia Community Additional supporting information may be found online in the Supporting Infor-
College, Alxandria, VA, USA mation section at the end of the article.
8 Faculty of Agricultural and Food
Sciences and Environmental
Management, University of
Debrecen, Hungary
9 Unidad de Investigación para la
Generación y Síntesis de
Evidencias en Salud,
Universidad San Ignacio de
Loyola, Lima, Peru
10 Centro de Investigación y
Transferencia de Tecnología -
CIITT, Universidad Católica de
Cuenca, Azogues, Ecuador
11 Translational Glycomics Group,
Research Institute of
Biomolecular and Chemical
Engineering, University of
Pannonia, Veszprém, Hungary

Received August 30, 2020

Revised October 4, 2020
Accepted October 27, 2020

Correspondence: Prof. Dr. Bence Mátyás, Ph.D., Genesis Sustain- 1 Introduction

able Future Ltd. 33 Rákóczi St., H-3950 Sárospatak, Hungary.
E-mail: bmatyas@genesissus.eu
Abbreviations: APTS, 8-aminopyrene-1,3,6-trisulfonic acid;
CE-LIF, capillary electrophoresis with laser induced fluores-
cence detection; FGs, free glycans; FNGs, free N-glycans; GC-
MS, gas chromatography–mass spectrometry; GU, Glucose
unit; PCT, plant complex type; SF-HPLC, size-fractionation
HPLC; TCI, triangular chlorophyll index
N-linked protein glycosylation is a topic of interest in plant
science [1–7] as it can modify various biological functions
[8,9], and affect growth, physiology, and development of liv-
ing organisms. N-glycans are linked to asparagine residues
in eukaryotic proteins [4]. Free forms of these glycans, free
N-glycans (FNGs), exist both in plants [4,10] and animals

© 2020 Wiley-VCH GmbH www.electrophoresis-journal.com

2 B. Mátyás et al.
[1,11,12], being produced by peptide N-glycanase (PNGase),
in a process, which involves an acidic PNGase (aPNGase) [4].
Free, non-conjugated N-glycans (PCT-FNGs) found in
various plants have been reported [1–7], however, their bio-
logical function has not yet been clarified. To the best of our
knowledge, these compounds are involved in salt stress tol-
erance for Arabidopsis [8]. Fanata et al. [9] reported that PCT-
FNGs play an essential role in several plant species. Hence,
in mutant rice, lack of PCT-FNGs caused reduced seedling
development; such plants died prior to reaching their repro-
ductive stage.
The most widespread methods for glycan analysis are liq-
uid separation techniques (CE, and LC-MS) and NMR [13].
Carbohydrate microarrays, having their solid surface modi-
fied with several different glycan structures, arranged in a
well-defined pattern, enable versatile and sensitive analysis
of altered glycosylation in complex biological samples [14].
Tsujimori and co-workers [15] used HPLC to assess the
presence of PCT-FNGs and several exoglycosidases in the
xylem sap of tomato plants. According to their study, PCT-
FNG derivatives are secreted and decomposed in the apoplast
fluid or xylem sap. Reily et al. [16] documented in human
studies that glycans play multiple crucial roles in cellular re-
sponse to environmental stimuli and in cellular growth and
differentiation, as well. Similar response can be observed in
plants, where specific changes in glycan composition can be
linked directly to nutrient deficiency, which represents a kind
of abiotic stress. In response, signal transduction pathways,
so called stress hormones (like ethylene, salicylic acid, jas-
monic acid, and abscisic acid) mediate the effect of the stress.
In nutrient deficiency, the concentration level of such hor-
mones rises. Kumar et al. [17] found two-fold higher sali-
cylic acid concentrations in cucumber seedlings, when no
nitrate supply was made available. Dubois and colleagues
[18] reported that nutrient deficiency induced ethylene syn-
thesis. Stress hormones trigger several mechanisms, such
as the induction of glycosyltransferases. Langlois-Meurinne
and coworkers [19] found that members of the D glycosyl-
transferases group had distinct induction profiles subsequent
to infection with Pseudomonas syringae pv in tomato or af-
ter treatment with salicylic acid or methyl jasmonate in Ara-
bidopsis. This finding indicates the potential role of glycosyl-
transferases in stress or defense response.
To establish whether quantified PCT-FNGs can serve as
markers of plant health status, we examined the relationship
between nutrient (nitrogen) deficiency and the types of com-
plex oligosaccharides. For evaluating plant health chlorophyll
content was determined via multispectral imaging. We quali-
fied possible PCT-FNGs in tomato xylem sap by capillary elec-
trophoresis with laser induced fluorescence detection (CE-
LIF). This is a sustainable technique, given that CE is suit-
able for miniaturization and has a low mass/power/volume
requirement relative to other liquid phase separation tech-
niques [20]. Due to scarcity of literature data, it was chal-
lenging to conduct this study. It must be noted, that struc-
tures found in databases originated from mammalian mon-
oclonal antibodies, rather than from plants. Even the gly-
© 2020 Wiley-VCH GmbH
Electrophoresis 2020, 0, 1–6
can database, Glycostore (https://glycostore.org) lists mainly
HPLC-based retention values, instead of CE-based ones. As
there are no available plant-related structures to serve as stan-
dards, we compared Glucose unit (GU) values obtained for
healthy and nutrient-deficient plant samples with GU values
listed in glycostore.org. We found that several peaks from
the CE profile of healthy crops are different from all peaks
present in the profiles of nutrient deficient crops. Assuming
that this finding is related to nutrient deficiency, we hypoth-
esized that possible glycan structures with peak differences
between healthy and nutrient deficient plants, are associated
with stress caused by nutrient (nitrogen) deficiency. Next, we
attempted to determine whether there is a universal norm
pertaining N-glycan functions, which can be related to nutri-
ent deficiency and/or diseases caused stress. To answer this
question, we scrutinized the literature of physiological pro-
cesses, for samples originating from either plants or eukary-
otes, in general.
As the initial step of our experimental work, we per-
formed the quantitative determination of nitrogen by Kjel-
dahl digestion. We found average total nitrogen content of
2.19 m/m% in nutrient deficient tomato samples and 4.30
m/m% in healthy tomato samples, respectively. A statistically
significant difference between the mean N content of healthy
and nutrient deficient plants: mean difference (healthy ver-
sus ND) was 2.1% and the p-value of the two-sided t-test was
0.0017.
2 Materials and methods
Multispectral analysis was performed in quadruplicate for
three tomato specimens per each group (healthy and nutri-
ent deficient plants) to determine the chlorophyll content.
Experiments were conducted on March 4, 2020, under natu-
ral sunlight, at Campus Sur of Universidad Politécnica Sale-
siana, in Quito, Ecuador. Nutrient deficient plants were se-
lected according to their symptoms visible to the naked eye.
The following warning signs were considered: (a) pale green
or yellowish spots on the upper leaf surface; (b) blackened
(necrotic) areas on the stems, (c) twisted and/or cupped up-
per foliage, and (d) expanded bronze areas.
Next, xylem sap was extracted from the same plant spec-
imens, on the same day.
2.1 Chemicals, reagents
Glacial acetic acid, sodium-cyanoborohydride (1 M in THF),
water (HPLC grade), and acetonitrile (99.8%) were purchased
from Sigma-Aldrich (St. Louis, MO, U.S.A). Fast Glycan La-
beling and Analysis Kit was obtained from SCIEX (Brea,
CA, U.S.A.) with a set of reagents included in the kit,
namely, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) tagging
dye, the maltooligosaccharide ladder, maltose, and magnetic
beads.
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Electrophoresis 2020, 0, 1–6
2.2 Chlorophyll and N determination
Total N was determined by the Kjeldahl method [21].
Multispectral images were captured by Parrot Sequoia
Multispectral Sensor (Parrot, Inc., France) to establish crop
health. Qgis 3.4 software was used to interpret multi-spectral
images. For calculating the Triangular Chlorophyll Index
(TCI) Eq. 1 was used, introduced by Haboudane et al. [22]:
1, 5 ∗ (R670 − R550 ) ∗ R700
TCI = 1, 2 ∗ (R700 − R550 ) − R670
2
(1)
where Rx is the light reflectance expressed in nm.
To realize possible nutrient deficiency in tomato plants,
the percentage values of chlorophyll captured by the sensor
were compared. A dimensionless value was obtained in the
range from −45 235 (low chlorophyll content) to 64 244 (high
chlorophyll content). Subsequently, chlorophyll percentages
were assigned based on seven color scales, as proposed by
Mendoza et al. [23]. Briefly, a pseudo color scale has been uti-
lized, from red to green, which identifies the areas with the
highest chlorophyll content, displayed in green (see Support-
ing Information). Chlorophyll values were compared with the
nitrogen concentration, using a SPAD-502 meter according
to Mendoza et al. [23].
2.3 Analysis of free oligosaccharides and
determination of their glucose unit values by
CE-LIF
2.3.1 Sample preparation
Sample preparation was done according to the protocol de-
scribed by Tsujimori et al. [15] and Masuda et al. [24]. In
brief, the stems of tomato seedlings were decapitated at ap-
proximately 5 cm above their root, using a previously steril-
ized stainless-steel razor blade. The first three drops of ex-
udate were discarded from the cut surface on the root side.
Cut surface was washed with distilled water and the exu-
date was collected as xylem sap with micropipettes (20-μL tip)
into ice-cooled tubes. Unlike Tsujimori and co-workers [14],
fresh sample solutions were analyzed. The obtained filtrate
(5–10 μL) was used for determining the expected free N type
oligosaccharides. All filtrates were freeze-dried in a vacuum
centrifuge at 30°C (at 2500 rpm, for approximately 1 h), until
the liquid evaporated completely from the samples. Then, the
free, non-conjugated oligosaccharides were labeled at their
reducing ends with a charged fluorophore, APTS, using the
Fast Glycan Labeling and Analysis kit from SCIEX [25]. After
labeling, the clean-up was performed according to the manu-
facturer’s protocol, i.e., samples were passed three times over
the magnetic beads included in the kit, to remove excess dye.
The charged fluorophore-labeled glycans were separated by
CE-LIF.
© 2020 Wiley-VCH GmbH
B. Mátyás et al. 3
Figure 1. Differences in average chlorophyll (%) index obtained
for 3 healthy and 3 nutrient deficient plant samples, respectively.
2.3.2 CE-LIF separation
APTS labeled filtrates were analyzed by means of a Proteome-
lab automated capillary electrophoresis system with laser in-
duced fluorescence detector module, equipped with a 520 nm
bandpass filter (Beckman-Coulter, Brea, CA, USA) [26]. Exci-
tation was done with a 405 nm diode laser (5.0 mW, Laser-
land, Wuhan, PRC). In all separations, a bare fused silica
(BFS) capillary was used (Polymicro Technologies, Phoenix,
AZ, USA), having 50 cm effective length (60 cm total length,
50 μm, ID). The cartridge temperature was set at 30°C for
all runs. The separation voltage was 30 kV, in reversed po-
larity mode (cathode at the injection side). Three-stage sam-
ple injection was utilized: (1) pressure-assisted water injec-
tion at 1 psi, for 5 s, (2) electrokinetic sample injection at
3 kV, for 3 s, and (3) APTS-labeled maltose (GU = 2) inter-
nal standard was injected at 1 kV, for 1 s. High resolution N-
linked Carbohydrate Separation gel buffer (HR-NCHO) from
the kit (SCIEX) served as the background electrolyte, and the
32Karat software package (version 9.2, SCIEX) was used for
data acquisition and processing. GU calculations were com-
pleted with GUcal v1.1B software (gucal.hu), with the internal
standard option as the basis of glycosylation analysis for all
sample components of interest (peaks) in the healthy and nu-
trient deficient electropherograms [27–31]. Relative percent-
age area values of the separated peaks were calculated by
means of PeakFit v4.12 software (SeaSolve Software Inc., San
Jose, CA). Further identification was based on consistent sta-
tistical methods, described in detail in Section 2.5.
3 Results and discussion
Differences in chlorophyll content between healthy and nu-
trient deficient tomato plants were also significant, as shown
in Fig. 1. In healthy samples the chlorophyll index varied be-
tween 90.00 and 96.54%, while in nutrient deficient plants we
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4 B. Mátyás et al. Electrophoresis 2020, 0, 1–6

Table 1. Comparison of relative peak areas within the ANOVA framework

Estimated mean 95% CI of the mean

a) )
No of pair difference difference p value (adjusted) GU

1 0.127 0.085; 0.170 <0.0001 4.19

2 0.085 0.037; 0.133 0.0148 4.92
4 0.118 0.075; 0.161 <0.0001
6 0.136 0.088; 0.184 <0.0001
7 0.124 0.081; 0.167 <0.0001
30 −0.116 −0.164; −0.069 0.0001 9.84

a)
Bonferroni-adjustment was applied.

observed values lower by almost one order of magnitude (in

the range from 11.99 to 12.04%).
As expected, a significant difference (p-value = 0.0053
estimated within the mixed model) was found between
the mean chlorophyll (henceforth: Cl) percent contained in
healthy (mean value of 95% CI) and nutrient deficient tomato
plants (32%). Chlorophyll indices are comparable those re-
ported by Mendoza and co-workers [23], who examined the
response to nitrogen content in tomato plants. Observed mul-
tispectral images and different chlorophyll percentages are
provided in Supporting Information. Table 1 and the IDs (the
serial number of pairs) showed on the X axis of Fig. 1 re-
veal that each chlorophyll percent-value of nutrient deficient
plants was lower than values recorded for healthy plants.
We identified 56 peaks for healthy tomato plant speci-
mens and 49 peaks from nutrient deficient tomato plants, re-
Figure 2. Comparative CE-LIF separation profiles of APTS labeled
spectively. All samples were analyzed in triplicate. 18 peaks
complex free, non-conjugated glycans. X-axis represents migra-
pairs (healthy; nutrient deficient) were selected for further tion times; y-axis displays Relative Fluorescent Unit values. Up-
analysis based on GU matches (Table 1). per trace: peaks of nutrient deficient tomato plant samples (ND).
From Fig. 2 and t-test results, we identified 6 peaks Lower trace: peaks of healthy tomato plant (H). Peak pairs in-
present in the profile of healthy crops as being different from cluded in the statistical analysis are numbered. Separation con-
ditions: APTS labeling, 50 cm (effective) BFS 50 um ID capillary,
all other peaks (in places with p value ≤ 0.0148; but mostly p
HR-NCHO separation gel buffer as BGE, and temperature of 30°C.
< 0.0001), resolved for nutrient deficient crops (Table 1). the Pressure-assisted water injection at 1 psi, for 5 s, then electroki-
authors believe that the presence of these structures in xylem netic sample injection at 3 kV, for 3 s, and APTS-labeled maltose
sap is associated with nutrient deficiency caused stress. internal standard was injected at 1 kV, for 1 s.
Lukacik et al. [32] stated that N-glycans are likely re-
sponsible for structure stabilizing and they commonly pro-
tect the main protein from stress-causing proteases. Also, of the GU value of 0.1 for 91% (≤0.2 for 97%), for neutral
Omtvedt and co-workers [33] detected the A2BG2 structure structures, and 0.2 for 95%, for charged structures. Our
and substantiated that glycan profiling can provide one addi- results are comparable to those reported by Campbell et al.
tional diagnostic opportunity in immunoglobulin-related dis- [35]; out of 105 possible structures analyzed, 91% had SD
eases [32,34]. Exoglycosidase-based sequencing should con- < 0.2. Therefore, to distinct oligosaccharide structures,
clude the structural match as part of a different study. two-sample t-tests with Satterthwaite correction were used,
which allowed to compare the mean of GU values of replicate
measurements on samples for all possible combinations of
3.1 Statistical analysis healthy and nutrient deficient plant peaks, at a two-sided 5%
significance level. No adjustment for multiplicity was made,
The aim of statistical analysis was to detect potential differ- as the simple null hypothesis of equal means was combined
ences between the profiles of healthy and nutrient deficient in a conjunctive manner into a composite null hypothesis,
plants, and to identify peaks in the electropherograms, which was rejected if and only if all simple null hypotheses
where the difference in CE separation of oligosaccharides could be rejected for a particular peak.
would occur. The approach described by Campbell et al. To compare relative peak areas, their values were first
[35] was applied to determine different peaks based on the logarithmized (i.e., logarithmically transformed via natural
SD of parallel measurements. The authors reported an SD logarithm), and then normalized to the total sum of all

© 2020 Wiley-VCH GmbH www.electrophoresis-journal.com

Electrophoresis 2020, 0, 1–6
log-transformed peak areas for each crop separately. Data
was pre-processed to identify the pair of peaks present for
both plant conditions (healthy and nutrient deficient). We
considered that one peak corresponds to a certain GU value
in both healthy and nutrient-deficient samples, if the follow-
ing condition was met. Only pairs with at least five resolved
peaks were included in the analysis; we defined as a complete
match, when the respective peak was present in at least five
electropherograms out of a total of six. Hence, the meaning
of match was that the peak could be detected in three healthy
samples and three nutrient-deficient plant samples, as well
or, alternatively in at least five of the six electropherograms.
These pairs of peak areas were then compared by con-
trasts within an ANOVA framework, accounting for plant
condition as a fixed effect, and the random factor of plant
nested within the condition. The Bonferroni correction was
applied for multiplicity, meaning that in order to preserve
the overall type I error rate of 0.05 the test-wise signifi-
cance level was divided by the total number of comparisons
performed.
The two conditions of the plant (healthy and nutrient de-
ficient) were also compared in terms of mean percentages of
chlorophyll content. A mixed model ANOVA was applied, ac-
counting for the fixed effect of condition and the random crop
effect (nested in condition).
Correlation between chlorophyll content and peak areas
could not be studied within a multivariate model, because
of the low number of crop samples (i.e., three for each con-
dition). Univariate Pearson-type correlations were computed
between the mean chlorophyll content of each specimen and
their normalized peak areas (only for peaks for which all six
data were available).
We compared each GU value originating from healthy
and nutrient deficient plant samples in which significant dif-
ferences between normalized peak areas were observed, with
structures that match our separation conditions (e.g., APTS
labeling, HR-NCHO separation gel buffer, separation temper-
ature 30°C, etc.). Considering that GU values are listed in the
Glycostore database up to two decimal places, we also con-
sidered matches at this degree of accuracy [36,37], but only
as a guide to follow for GU accuracy, rather than for struc-
tural comparison. All calculations were performed with SAS
version 9.4.
4 Concluding remarks
Statistically significant, negative correlation between chloro-
phyll content and normalized peak areas was found for four
peaks, as shown in Table 2.
We revealed a statistical correlation between nutrient-
deficient symptoms of crops (predicted by multispectral
images and determination of total nitrogen content) and
changes in suspected free, non-conjugated N-glycans (PCT-
FGs) profiles (based exclusively on GU values, determined
by CE-LIF alone). Therefore, we intend to further investigate
how specific these findings were. Beyond doubt, our results
© 2020 Wiley-VCH GmbH
B. Mátyás et al. 5
Table 2. Pearson correlation between chlorophyll content and
peak areas (statistically significant results only, ordered
by the extent of correlation)
No of pair Correlation p-value
1 −0.896 0.0157
4 −0.888 0.0182
3 −0.886 0.0189
2 −0.815 0.0482
suggest that changes in free glycan profiles detected in xylem
sap represent an early indicator of plant disease. Hence, CE-
LIF should prove useful for determining the number and ra-
tio of N-glycans in plant samples.
Limitation of the study: For rapid detection of nutrient de-
ficiency in plants, we used multispectral imaging, as stated in
the Methods section. Nutrient deficient plants were selected
according to their symptoms visible to the naked eye.
4.1 Recommendations
Future studies should be complemented by genomic tests
for different plant diseases and/or nutrient deficiency (in
this case shortage in N) to validate multispectral data prior
to further analyses. Methods used in the study (multispec-
tral analysis and capillary electrophoresis with laser induced
fluorescence detection) enabled sustainable measurements
and novel technological solutions. Exoglycosidase digestion
based structural confirmation was beyond the scope of this
study.
Given the significant correlation found between GU val-
ues and crop health, the authors will continue working on
large scale glycan-profiling of plant samples, and will make
their results available to the scientific community in an open-
access database, named glycoplants.org (pre-launched).
The authors express special thanks to all members of the
Biotechnology Engineering of Natural Resources, Universidad
Politécnica Salesiana, for their assistance in sample preparation.
Authors are indebted to Mr. David Loja (Agroscan); and Prof. Dr.
Lenin Ramirez for multispectral imaging, and initial discussions
on the choice of statistical methods. Authors gratefully acknowl-
edge support by Genesis Sustainable Future Ltd., Hungary; Dama
Research Center limited, Hong Kong, and BIONANO_GINOP-
2.3.2-15-2016-00017 project and the V4-Korea Joint Research
Program, project National Research, Development and Innova-
tion Office (NKFIH) (NN 127062), both grants provided by the
Hungarian Government. The authors also benefitted of EFOP-
3.6.3-VEKOP-16-2017-00009, co-financed by EU and the Euro-
pean Social Fund. This is contribution #178 made by the Horváth
Csaba Memorial Laboratory of Bioseparation Sciences. The au-
thors thank Ms. Alice A. Lowy for proofreading the manuscript
and giving useful suggestions.
The authors have declared no conflict of interest.
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6 B. Mátyás et al.
Data availability statement
All figures and tables in the Supporting Information are as-
sociated with raw data.
5 References
[1] Strasser, R., Front. Plant Sci. 2014, 5, 363.
[2] Wilson, I. B. H., Zeleny, R., Kolarich, D., Staudacher, E.,
Stroop C. J. M., Kamerling, J. P., Altmann, F., Glycobiol-
ogy 2001, 11, 261–274.
[3] Arigoni-Affolter, I., Scibona, E., Lin, C.-W., Brühlmann,
D., Souquet, J., Broly, H., Aebi, M., Sci. Adv. 2019, 5,
eaax8930.
[4] Maeda, M., Kimura, Y., Front. Plant Sci. 2015, 5, 429.
[5] Kimura, Y., Kitahara, E., Biosci. Biotechnol. Biochem.
2000, 64, 1847–1855.
[6] Kimura, Y., Matsuo, S., J. Biochem. 2000, 127, 1013–1019.
[7] Maeda M., Kimura, M., Kimura, Y., J. Biochem. 2010, 148,
681–692.
[8] Kang, J. S., Frank, J., Kang, C. H., Kajiura, H., Vikram, M.,
Ueda, A., Kim, S., Bahk, J. D., Triplett, B., Fujiyama, K.,
Lee, S. Y., von Schaewen, A., Koiwa, H., Proc. Natl. Acad.
Sci. U. S. A. 2008, 105, 5933–5938.
[9] Fanata, W. I. D., Lee, K. H., Son, B. H., Yoo, J. Y., Harmoko,
R., Ko, K. S., Ramasamy, N. K., Kim, K. H., Oh, D.-B., Jung,
H. S., Kim, J.-Y., Lee, S. Y., Lee, K. O., Plant J. 2013, 73,
966–979.
[10] Maeda, M., Ebara, N., Tani, M., Vavricka, C. J., Kimura,
Y., Glycoconjugate J. 2017, 34, 229–240.
[11] Karav, S., Casaburi, G., Arslan, A., Kaplan, M., Sucu, B.,
Frese, S., J. Functional Foods 2019, 61, 103485.
[12] Seino, J., Fujihira, H., Shin-ichi Nakakita, S.-I., Masahara-
Negishi, Y., Miyoshi, E., Hirabayashi, J., Suzuki, T., Gly-
cobiology 2016, 26, 1072–1085.
[13] Mulloy, B., Hart, G. W., Stanley, P., in: Varki A, Cum-
mings, R. D., Esko, J. D., Stanley, P., Hart, G. W., Aebi,
M., Darvill, A. G., Kinoshita, T., Packer, N. H., Prestegard,
J. H., Schnaar, R. L., Seeberger, P. H. (Eds.), Essentials
of Glycobiology, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY 2017, pp.2015–2017.
[14] Donczo, B., Kerekgyarto, J., Szurmai, Z., Guttman, A.,
Analyst 2014, 139, 26502657.
[15] Tsujimori, Y., Ogura, M., Rahman, M. Z., Maeda, M.,
Kimura, Y. Biosci. Biotech. Biochem. 2019, 83, 1310–1314.
[16] Reily, C., Stewart, T. J., Renfrow, M. B., Novak, J., Nat.
Rev. Nephrol. 2019, 15, 346–366.
[17] Kumar, S. P., Kumar, C. V., Bandana, B., J. Stress Physiol.
Biochem. 2010, 6, 102–113.
[18] Dubois, M., Van den Broeck, L., Inzé, D., Trends Plant Sci.
2018, 23, 311–323.
© 2020 Wiley-VCH GmbH
Electrophoresis 2020, 0, 1–6
[19] Langlois-Meurinne, M., Gachon, C. M., Saindrenan, P.,
Plant Physiol. 2005, 139, 1890–1901.
[20] Mátyás, B., Bautista, G., Szarka, M., Serrano, V., Morales
Arteaga, J., Loja, D., Yaguana, S.G., Gómez, F., Ramírez-
Cando, L. J., Electrophoresis 2018, 39, 2884–2889.
[21] Kirk, P. L., Anal. Chem. 1950, 22, 354–358.
[22] Haboudane, D., Tremblay, N., Miller, J. R., Vigneault,
P., IEEE Trans. Geosci. Remote Sens. 2008, 46,
423–437.
[23] Mendoza, M. D. L. N. R., González, G. A., Santelises, A.
A., Barra, J. D. E., Rincón, J. A. S., Terra Latinoamericana
1998, 16, 135–141.
[24] Masuda, S., Sakuta, C., Satoh, S., Plant Cell Physiol.
1998, 39, 1330–1336.
[25] SCIEX the Power of Precision, Fast Glycan La-
beling and Analysis Kit: Application Guide,
https://SCIEX.com/products/consumables/fast-glycan-
analysis-and-labeling-for-the-pa-800-plus (accessed:
July 2020).
[26] Beckman-Coulter, PA 800 Plus Pharmaceutical Analysis
System—Sciex, https://sciex.com/Documents/manuals/
PA-800-Plus-Maintenance-Guide.pdf (accessed: July
2020).
[27] Szigeti, M., Bondar, J., Gjerde, D., Keresztessy, Z.,
Szekrenyes, A., Guttman, A., J. Chromatogr. B 2016,
1032, 139–143.
[28] Szigeti, M., Guttman, A., Mol. Cell. Proteomics 2019, 18,
2524–2531.
[29] Jarvas, G., Szigeti, M., Guttman, A., J. Proteomics 2018,
171, 107–115.
[30] Jarvas, G., Szigeti, M., Guttman, A., Electrophoresis
2015, 36, 3094–3096.
[31] DRC Sustain. Future 2020, (1), 60.65.
[32] Lukacik, P., Roversi, P., White, J., Esser, D., Smith, G. P.,
Billington, J., Williams, P. A., Rudd, P. M., Wormald, M.
R., Harvey, D. J., Crispin, M. D., Radcliffe, C. M., Dwek,
R. A., Evans, D. J., Morgan, B. P., Smith, R. A., Lea, S. M.,
Proc. Nat. Acad. Sci. U. S. A. 2004, 101, 1279–1284.
[33] Omtvedt, L. A., Royle, L., Husby, G., Sletten, K., Radcliffe,
C. M., Harvey, D. J., Dwek, R. A., Rudd, P. M., Arthritis
Rheumatol. 2006, 54, 3433–3440.
[34] Watson, M., Rudd, P. M., Bland, M., Dwek, R. A., Axford,
J. S., Arthritis Rheumatol. 1999, 42, 1682–1690.
[35] Campbell, M. P., Royle, L., Radcliffe, C. M., Dwek, R. A.,
Rudd, P. M. Bioinformatics 2008, 24, 1214–1216.
[36] GU Database, Lendület III - MTA-PE Translational Gly-
comics, Universitas Pannonica, https://lendulet.uni-
pannon.hu/index.php/gudatabase (accessed: July
2020).
[37] GlycoStore, https://glycostore.org/glycan/624
(accessed: July 2020).
www.electrophoresis-journal.com