Selective detection of carbohydrates and their peptide conjugates by esi-ms using synthetic quaternary ammonium salt derivatives of Phenylboronic acids

Article abstract of DOI:10.1007/s13361-014-0857-4

We present new tags based on the derivatives of phenylboronic acid and apply them for the selective detection of sugars and peptide-sugar conjugates in mass spectrometry. We investigated the binding of phenylboronic acid and its quaternary ammonium salt (QAS) derivatives to carbohydrates and peptide-derived Amadori products by HR-MS and MS/MS experiments. The formation of complexes between sugar or sugar-peptide conjugates and synthetic tags was confirmed on the basis of the unique isotopic distribution resulting from the presence of boron atom. Moreover, incorporation of a quaternary ammonium salt dramatically improved the efficiency of ionization in mass spectrometry. It was found that the formation of a complex with phenylboronic acid stabilizes the sugar moiety in glycated peptides, resulting in simplification of the fragmentation pattern of peptide-derived Amadori products. The obtained results suggest that derivatization of phenylboronic acid as QAS is a promising method for sensitive ESI-MS detection of carbohydrates and their conjugates formed by non-enzymatic glycation or glycosylation. [Figure not available: see fulltext.]

Full text of DOI:10.1007/s13361-014-0857-4

B The Author(s), 2014. This article is published with open access at Springerlink.com
J. Am. Soc. Mass Spectrom. (2014) 25:966Y976
DOI: 10.1007/s13361-014-0857-4
RESEARCH ARTICLE
Selective Detection of Carbohydrates and Their Peptide
Conjugates by ESI-MS Using Synthetic Quaternary
Ammonium Salt Derivatives of Phenylboronic Acids
Monika Kijewska,1 Adam Kuc,1 Alicja Kluczyk,1 Mateusz Waliczek,1
Aleksandra Man-Kupisinska,2 Jolanta Lukasiewicz,2 Piotr Stefanowicz,1
Zbigniew Szewczuk1
1Faculty of Chemistry, University of Wroclaw, Wroclaw, Poland
2Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy
of Sciences, Wroclaw, Poland
Abstract. We present new tags based on the derivatives of phenylboronic acid
and apply them for the selective detection of sugars and peptide-sugar conjugates
in mass spectrometry. We investigated the binding of phenylboronic acid and its
quaternary ammonium salt (QAS) derivatives to carbohydrates and peptide-
derived Amadori products by HR-MS and MS/MS experiments. The formation of
complexes between sugar or sugar-peptide conjugates and synthetic tags was
confirmed on the basis of the unique isotopic distribution resulting from the
presence of boron atom. Moreover, incorporation of a quaternary ammonium salt
dramatically improved the efficiency of ionization in mass spectrometry. It was
found that the formation of a complex with phenylboronic acid stabilizes the sugar
moiety in glycated peptides, resulting in simplification of the fragmentation pattern of peptide-derived
Amadori products. The obtained results suggest that derivatization of phenylboronic acid as QAS is a
promising method for sensitive ESI-MS detection of carbohydrates and their conjugates formed by non-
enzymatic glycation or glycosylation.
Key words: Quaternary ammonium salt, Mass spectrometry, Solid phase synthesis, Amadori products, Non-
enzymatic glycation, Sugars, Phenylboronic acids
Received: 3 January 2014/Revised: 9 February 2014/Accepted: 15 February 2014/Published Online: 1 April 2014
modifications by conventional mass spectrometry methods.
The MS analysis is also affected by the insufficient
Introduction
ionization efficiency of these peptides. The problem of
selective detection of post-translational modifications, in-
cluding glycation, was widely discussed in literature [3–9].
The direct sequencing of the peptide-derived Amadori
products by collision induced dissociation (CID) is non-
efficient because the CID spectrum of a glycated peptide
reveals the characteristic pattern of neutral losses (several
molecules of water, formaldehyde, and finally the whole
hexose moiety) [3–5]. The abundance of peptide backbone
fragments is relatively low and the sequence coverage is not
sufficient to reconstruct the sequence of the studied peptide.
Therefore, new techniques of ECD and ETD have been
applied for the sequencing of the Amadori products [6, 7].
Another approach to the analysis of the primary structure of
these compounds was based on a combination of enzymatic
hydrolysis with mass spectrometry [8, 9]. Because of a very
lycation, one of the post-translational modifications of
proteins, is a nonenzymatic reaction between sugar
G
aldehyde or ketone and the amino groups of proteins. In the
early stage of glycation, the intermediates leading to the
Amadori compounds are formed [1]. Glucose-induced
damage is not limited to diabetic patients; glycation also
affects physiological aging and neurodegenerative diseases
such as Alzheimer's disease and amyotrophic lateral sclero-
sis [2]. The modifications are formed at very low concen-
trations, which limit detection sensitivity of these
Electronic supplementary material The online version of this article
(doi:10.1007/s13361-014-0857-4) contains supplementary material, which
is available to authorized users.
Correspondence to: Monika Kijewska; e-mail: monika.kijewska@chem.uni.wroc.pl

M. Kijewska et al.: Phenylboronic QAS Tags for Carbohydrates
967
low concentration of modified proteins and peptides, new
methods of selective detection based on isotopic labeling
(13C or 18O) combined with mass spectrometry were
researched [10, 11]. Another approach to overcome the
low concentration of analyzed compounds is the application
of sample preconcentration, frequently based on affinity
methods. In case of LC-MS analysis of glycated peptides,
borate affinity columns based on the specific interaction
between phenylboronic acid derivatives and diols in sugar
moieties have been used [12, 13].
It has been known for nearly 50 years that borate ion will
complex with diols in solution forming borate esters [14].
The polyol structures of carbohydrates in glycopeptides can
reversibly form anionic borate esters in the presence of
tetrahydroxyborate ion, B(OH4)– at alkaline pH (pH 8–10),
which might be effectively used to distinguish glycopeptides
from peptides [15]. The use of this reaction in combination
with CE and HPLC has been reported for the separation of
individual oligosaccharides released from glycoproteins and
peptides with glycan variations, carbohydrates, and Amadori
products [16–18]. Moreover, Chen and coworkers have
demonstrated that surface plasmon resonance (SPR) can
successfully detect and quantify the binding of HbA1c to
phenylboronic acid monolayer by the specific interaction.
This methodology may prove to be useful in clinical
diagnosis and serve as an assay for HbA1c binding ability
[19].
The reversible covalent association of phenylboronic
acids and 1,2-cis-diols, catechols, or 2-hydroxycarboxylates
to form cyclic boronic esters is one of the most explored
interactions in molecular recognition [20]. In combination
with salt bridges and hydrogen bonding, markers based on
boronic acids have been developed for citrate [21], glucose-
6-phosphate [22], and heparin [23]. Recently, a synthetic
library of simple bis-boronic acid-based receptors with
various spacers was used for the sensing of ginsenosides.
The incorporation of two boronic acids allowed the pairing
of two indicators, which can simultaneously bind the
receptors or two saccharides within the ginsenosides.
Moreover, the assay reported should be applicable to the
analysis of other large saccharide-based natural products
[24].
analysis of isomeric polyols using negative ion ESI-MS
in conjunction with CID of the complexes [31]. Nega-
tive-ion-mode ESI-MS/MS is widely used for the
analysis of the structures of borate complexes with
different carbohydrates [32, 33]. In our recent work, we
proposed an alternative method for selective detection of
glycated peptides by mass spectrometry based on the
borate complexes [34]. We presented the experiments on
the formation and CID of borate complexes of peptide-
derived Amadori products. MS experiments were per-
formed in positive ion mode, which greatly facilitated the
analysis of the MS/MS spectra. Our results indicate that
the complexation of the borate anion by a glycated
peptide may be used to distinguish glycated and non-
glycated peptides; moreover, the complex stabilizes the
hexose moiety attached to the lysine side chain, which
reduces the neutral losses and simplifies CID spectrum
[34].
In this work, we expanded our research on interac-
tions of peptide-derived Amadori products and carbohy-
drates with phenylboronic acid and phenylboronic acid
derivatives. We designed and synthesized new tags based
on the combination of phenylboronic acid and quaternary
ammonium salt and used them for selective detection of
sugars and sugar-peptide conjugates, taking into account
that incorporation of QAS into peptide chain significantly
improves the efficiency of ionization of these compounds
by mass spectrometry [35]. We performed the ESI-MS
and ESI-MS/MS experiments using the model glycated
peptides and biological samples to confirm the applica-
bility of the selective detection method presented in this
work. Our results indicate that complexes of glycated
peptides and phenylboronic acid or phenylboronic acid
derivatives also stabilize the sugar moiety in CID
experiments. It could be expected that application of
the new tags may improve selective and sensitive
detection of carbohydrates or polysaccharides in positive
ion mode in mass spectrometry.
Experimental
Reagents
Although there is much written in the literature on the
investigation of structures of borate polyol complexes by
nuclear magnetic resonance spectroscopy (NMR) [25–28]
and potentiometry [28], particularly with carbohydrates,
there have been only a few reports on the use of mass
spectrometry to investigate borate complexes. Two
different techniques for the ionization of sample, fast
atom bombardment (FAB) and negative ion electrospray
(ESI), were investigated to confirm a number of boronic
acid molecules, both showing excellent results [29].
Negative ion-mode FAB-MS was also used for the
analysis of the structure of methyl β-D-apiofuranoside–
borate complex [30]. Ackloo et al. have applied the
concept of boric acid complexation to explore structural
All solvents and reagents were used as supplied. Fmoc
amino acid derivatives were purchased from
NovaBiochem (as Merck, Darmstadt, Germany). O-(6-
chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tet-
rafluoroborate (TCTU), the MBHA-Rink amide resin
(0.67 mmol/g), and trifluoroacetic acid (TFA) were
obtained from IrisBiotech (Marktredwitz, Germany). DABCO
(1,4-diazabicyclo[2.2.2]octane), 4-(dimethylamino)phenylboronic
acid, and 4-carboxyphenylboronic acid for the synthesis
of quaternary ammonium salts (QAS) and solvents for
peptide synthesis: N,N-dimethylformamide (DMF), di-
chloromethane (DCM), and N-ethyldiisopropylamine
(DIEA) were obtained from Sigma Aldrich (St. Louis,

968
M. Kijewska et al.: Phenylboronic QAS Tags for Carbohydrates
MO, USA); iodoacetic acid from Merck (Darmstadt, Germany);
N,N′-diisopropylcarbodiimide (DIC) and triisopropylsilane (TIS)
from Fluka (as St. Louis, MO, USA). HPLC grade solvents used
for purification [acetonitrile (MeCN) and methanol (MeOH)]
were from Sigma Aldrich. Carbohydrates were obtained from
Prolabo (Gdańsk, Poland) (D-arabinose), Fluka (D-ribose),
ChemPur (D-glucose), Windsor Laboratory (D-glucuronic acid),
Fluka (D-2-deoxyribose), Sigma-Aldrich (N-acetylglucosamine),
Fluka (saccharose). Ammonium carbonate was purchased from
Sigma Aldrich, the pH of the 50 mM buffer was adjusted to 9.0
with ammonia solution.
The oligosaccharide of lipopolysaccharide containing one
RU linked to Hep-Kdo disaccharide was isolated from Hafnia
alvei (Polish Collection of Microorganisms) PCM 1200
lipopolysaccharide (LPS) in the Department of Immunochem-
istry, Ludwik Hirszfeld Institute Immunology and Experimen-
tal Therapy, Polish Academy of Sciences [36].
resin (30 mg, 0.02 mmol) was washed with DCM (3 × 1 min),
DCM/DMF (1:1; vol:vol, 1 min), 5% DIEA in DMF (3 × 1 min),
and DMF (7 × 1 min). The mixture of iodoacetic acid (18.7 mg,
0.1 mmol) and DIC (15.6 μL 0.1 mmol) dissolved in DMF
(0.5 mL) was added and the reaction was allowed to proceed for
90 min with exchange of reagents solution after each 30 min.
Then DABCO (45.1 mg, 0.4 mmol), dissolved in DMF (0.5 mL)
was added to the reaction vessel and mixed for 12 h. After
completing the synthesis of the QAS, the N-terminal Fmoc group
was removed using the 25% solution of piperidine in DMF, and
4-carboxyphenylboronic acid (10 mg, 0.06 mmol) was attached,
using TCTU (21 mg, 0.06 mmol) and DIEA (21 μL, 0.12 mmol)
for 2 h at room temperature. The product was cleaved from the
resin with the mixture of TFA/TIS/H2O (95:2.5:2.5; vol:vol:vol)
for 2 h at room temperature and precipitated with cold diethyl
ether (see Figure 1).
Yield: 52%, HPLC: Rt (min): 11.9 (conditions for HPLC
are given in the Experimental section, Purification).
HR-MS (ESI-FT): m/z: [M]+ Calcd. for C21H33N5O5B
446.256; Found: 446.257.
Glycated Peptides
The mono- and diglycated fragments of BSA [H-Asp-Thr-
Glu-Lys-Gln-Ile-Lys(Fru)-Lys-Gln-Thr-OH, H-Asp-Thr-
Glu-Lys(Fru)-Gln-Ile-Lys(Fru)-Lys-Gln-Thr-OH] and the
model peptide H-Ala-Lys(Fru)-Ala-Phe-OH were prepared
on solid support using the novel lysine derivative (Nα-9-
fluorenylmethoxycarbonyl-Nε-tert-butyloxycarbonyl-Nε-
(2,3:4,5-di-O-isopropyliden-1-deoxy-β-D-fructopyranose-1-
ylo)lysine, Fmoc-Lys(i,i-Fru,Boc)-OH. The details of the
synthetic procedure were given in our previous paper [8].
Fmoc-Lys(Fru)-OH (Nα-9-fluorenylmethoxycarbonyl-Nε-
(1-deoxy-β-D-fructopyranose-1-ylo)lysine) was obtained by
deprotection of Fmoc-Lys(i,i-Fru,BOC)-OH with TFA con-
taining 5% of water. After 8 h, TFA was evaporated and the
reaction product was used for MS experiments without
further purification.
Synthesis of PhB(QAS)-GGG-NH2 (TAG2): Peptides
were prepared on solid support, using the Rink resin and
standard Fmoc synthetic procedure. After synthesis of
Fmoc-Gly-Gly-Gly-Rink, the α-amino group of glycine
residue was derivatized on solid support using 4-
(dimethylamino)phenylboronic acid to form QAS. The
Fmoc protecting group was removed by using 25% solution
of piperidine in DMF (1 × 3 min, 1 × 17 min). After the
peptidyl resin (30 mg, 0.02 mmol) was washed with DMF
(7 × 1 min), the mixture of iodoacetic acid (18.7 mg,
0.1 mmol) and DIC (15.6 μL, 0.1 mmol), dissolved in DMF
(0.5 mL), was added and the reaction was allowed to
proceed for 90 min with exchange of reagents solution after
each 30 min. Then 4-(dimethylamino)phenylboronic acid
(66.3 mg, 0.4 mmol), dissolved in DMF (0.5 mL), was
added to the reaction vessel and mixed for 24 h. After
incubation, the peptidyl resin was washed with DMF
(7 × 1 min) and the product (PhB(QAS)-GGG-NH2) was
cleaved from the resin using TFA/H2O/TIS (95:2.5:2.5, vol/
vol) for 2 h at room temperature and precipitated with cold
diethyl ether (see Figure 2).
Preparation of the Hydrolysate of Glycated
Ubiquitin
The glycation of ubiquitin followed by the enzymatic
hydrolysis of the glycation product were performed accord-
ing the procedures described previously [6].
Yield: 96%, HPLC: Rt (min): 7.1 (conditions for HPLC
are given in the Experimental section, Purification).
HR-MS: (ESI-FT): m/z: [M]+ Calcd. for C16H25N5O6B
394.190; Found: 394.189.
Synthesis of Derivatives of Phenylboronic Acid
and QAS
Products were prepared by manual solid-phase synthesis
techniques, using the standard Fmoc synthetic procedure.
The methodology of derivatization of lysine residues by
DABCO in peptides on solid support was similar to the one
presented by us previously [37].
Synthesis of PhB-K(QAS)-NH2 (TAG1): After attaching
Fmoc-Lys(Mtt)-OH to the Rink resin, the Mtt protecting group
was removed by using 1% solution of TFA in DCM (3 × 2 min, 3
× 10 min, 3 × 2 min) and the ε-amino group of the lysine residue
was derivatized on solid support to form QAS. Then the peptidyl
Purification
The crude products (QAS-phenylboronic acid derivatives)
were analyzed using a Thermo Separation HPLC system
with a UV detection (210 nm) on a Vydac Protein RP C18
column (4.6 × 250 mm, 5 μm), with a gradient elution of
0%–80% B in A (A = 0.1% TFA in water; B = 0.1% TFA in
acetonitrile/H2O, 4:1) for 40 min (flow rate 1 mL/min). The
main reaction product was purified by a preparative
reversed-phase HPLC on a Tosoh (Tosoh Tokyo, Japan)

M. Kijewska et al.: Phenylboronic QAS Tags for Carbohydrates
969
TSKgel with an ODS-120 T column (21.5 mm × 300 mm),
For MS spectra analysis, a Bruker Compass DataAnalysis
using a linear gradient 10%–20% of B for 40 min (PhB-
K(QAS)-NH2) and 2%–12% of B for 40 min (PhB(QAS)-
GGG-NH2), flow rate 7.0 mL/min, UV detection at 220 nm.
The fractions were collected and lyophilized. The identities
of the products were confirmed by MS analysis using Bruker
Apex-Qe (Bremen, Germany) Ultra 7 T FT-ICR mass
spectrometer equipped with an electrospray ionization
source.
4.0 software was used. A sophisticated numerical annotation
procedure (SNAP) algorithm was used for finding peaks. All
obtained signals had mass accuracy error in the range of
5 ppm. Spectra (positive ion mode) were recorded using a
50:50 acetonitrile–water mixture containing 0.1% HCOOH
for standard analysis (e.g., confirmation of structure of
obtained peptides and tags).
Results and Discussion
Complex Formation
Complex Formation Using Phenylboronic Acid
Samples of carbohydrate mixtures, Fmoc-Lys(i-Fru)-OH,
glycated peptides, glycated ubiquitin hydrolysate, or core
oligosaccharide were prepared in water with final concen-
tration of 1 mg/mL. The aliquots (10–20 μL) were diluted by
50 mM ammonium carbonate buffer (500 μL). Then, 5 μL
of phenylboronic acid solution (PhB, 1 mg in 1 mL of
MeOH) or phenylboronic acid derivatives – 2 μL PhB-
K(QAS)-NH2 (TAG1)(0.6 mg in 1 mL of H2O) or 3 μL
PhB(QAS)-GGG-NH2 (TAG2)(0.6 mg in 1 mL of H2O) was
added. After 1 min incubation, the sample was diluted with
methanol (1:1, vol:vol) and subjected to ESI-MS experi-
ment. All samples were also analyzed in ammonium borate
buffer methanol mixture without complexing reagents.
The series of model synthetic derivatives (glycated
peptides and Fmoc-Lys(Fru)-OH) as well as peptic
fragments of ubiquitin were tested for their affinity
towards the phenylboronic acid. In our previous work,
we performed similar experiments to examine the stability
of complexes of Amadori products with borate ion in
mass spectrometry. We showed that the complexes are
stable in the basic (pH 99) water-methanol solution [34].
Moreover, the attachment of borate ion to the
fructosamine moiety stabilizes the sugar-bearing side
chain in glycated peptides, which simplifies the analysis
of CID spectra. The distance between the peak corre-
sponding to the glycated peptide and its borate complex is
only 8 Da, which may complicate interpretation of
multiply charged ions because of the overlap of signals.
To facilitate the interpretation of mass spectra, we used
phenylboronic acid for the complex formation. The mixture
of Fmoc-Lys(Fru)-OH and phenylboronic acid was incubat-
ed in ammonium carbonate buffer at the pH 9.0 (the details
are included in Experimental). After 1 min the
sample was diluted with methanol and analyzed using ESI-
MS. The MS spectrum presented in Figure 3 shows a series of
complexes containing phenylboronate anion. The most abun-
dant complex contains one phenylboronate anion and one
molecule of Fmoc-Lys(Fru)-OH, however, a complex contain-
ing two phenylboronate ions was also observed. The
stoichiometry of these complexes is proven by their isotopic
patterns. The parent ions at m/z 531.234, 617.269, and
703.304 were subjected to CID experiments (Supplemental
Figures S1, S2, S3). The CID experiments were performed at
the same range of collision energy of 5–35 eV to check the
stability of the formed complex. In the MS spectrum of free
glycated lysine, the characteristic neutral losses are observed
(Supplemental Figure S1). The fragmentation of the peak
corresponding to the complex of Fmoc-Lys(Fru)-OH with
phenylboronic acid is characterized by a low abundance
of ions formed by the elimination of water, but the ion
formed by formaldehyde elimination is observed, although
only after elimination of phenylboronate ion. In
this case, the elimination of phenylboronate ion could be
observed in mass spectrometry experiments (ion 447.193
Mass Spectrometric Measurements
The samples were analyzed using micrOTOF-Q and Apex-
Qe Ultra 7 T mass spectrometers (Bruker Daltonics). The
micrOTOF-Q instrument equipped with an ESI source with
ion funnel, was operated in positive or negative ion mode
and calibrated before each analysis with the Tunemix
mixture (Bruker Daltonics) in a quadratic method. In MS/
MS experiments, the collision energy (5–40 eV) was
optimized for the best fragmentation. Argon was used as a
collision gas. The spectra were recorded in ammonium
carbonate buffer (pH 9.0) at the peptide concentration of
0.5 μM.
The FT-ICR (Fourier transform ion cyclotron resonance)
Apex-Qe Ultra 7 T instrument, equipped with an ESI source,
was operated in the positive or negative ion mode. Analyte
solutions were introduced at a flow rate of 3 μL/min. The
instrument was calibrated before each analysis with the
Tunemix mixture (Bruker Daltonics) in a quadratic method.
The instrument parameters were as follows: scan range:
100–1600m/z; drying gas: nitrogen; temperature: 200ºC;
potential between the spray needle and the orifice: 4.5 kV. In
the MS/MS experiments, precursor ions were selected on the
quadrupole and subsequently fragmented in the hexapole
collision cell. Argon was used as a collision gas. Obtained
fragments were directed to the ICR mass analyzer and
registered as an MS/MS spectrum. The collision voltage was
optimized for the best fragmentation pattern.

970
M. Kijewska et al.: Phenylboronic QAS Tags for Carbohydrates
Figure 1. Synthesis of PhB-K(QAS)-NH2
m/z – Supplemental Figure S2). In our previous
research, the collision energy required to obtain charac-
teristic neutral losses for complex Fmoc-Lys(Fru)-OH
with borate ion was lower [34].
Figure S5). The fragmentation of this ion was compared
with results obtained for the protonated free glycated
peptide (published previously [34]). The CID experiments
were performed at a range of the collision energy of 5–
45 eV to check the stability of the formed complex. The
fragmentation spectrum of the complex of the model
glycated peptide with phenylboronate is characterized by
low abundance of ions formed by the elimination of
water, and a low abundance peak corresponding to the ion
formed by the elimination of a whole hexose moiety is
observed (m/z 436.265). However, the fragmentation
spectrum is dominated by the series of b and y ions
covering the whole sequence of the peptide. In our
previous work, focused on the study of stability of
modified peptide–borate complex, the elimination of
formaldehyde and a whole hexose moiety was not
The model glycated peptide H-Ala-Lys(Fru)-Ala-Phe-OH
was also incubated with phenylboronic acid. The represen-
tative MS spectrum for the complex of this glycated peptide
and phenylboronic acid is shown in Supplemental
Figure S4. The peak at m/z 598.31 corresponds to the
protonated peptide H-Ala-Lys(Fru)-Ala-Phe-OH, whereas
the proposed structure for the ion at m/z 684.35 is
presented in the Supplemental Figure S4. The m/z value
and the characteristic isotopic pattern for this signal are in
good agreement with the molecular formula
C33H46N5O10B+. The parent ion at m/z 684.35 was
subjected to MS/MS fragmentation (Supplemental

M. Kijewska et al.: Phenylboronic QAS Tags for Carbohydrates
971
Figure 2. Synthesis of PhB(QAS)-GGG-NH2
observed. We attributed the stability of the obtained
complex to the stabilization of aminofructose moiety,
which may be related to the crosslinking of the hexose
with formation of a multi-ring structure [34] and the
subsequent decrease in basicity (and, consequently, the
protonation level) of the nitrogen attached to hexose
moiety [38]. The analysis of the spectrum of the H-Ala-
Lys(Fru)-Ala-Phe-OH peptide complexed with
phenylboronic acid revealed that only two hydroxyl
groups are involved in phenylboronic complexes, which
may explain the lower collision energy required to
fragment and eliminate the sugar moiety. Although the
stabilization of hexose moiety is lower, it is still sufficient
to indicate the modification site.
The selective detection of glycated peptides was studied
on the glycated ubiquitin hydrolysate. The whole peptic
digest was incubated with phenylboronic acid in ammoni-
um carbonate buffer, diluted with methanol, and directly
analyzed on a micrOTOF instrument. The obtained results
are presented in Figure 4 (the expanded range 600–825m/z
of obtained spectrum). The glycated peptides were identi-
fied on the basis of their characteristic isotopic pattern,
comparing the spectra of the peptic mixture recorded
without additives or in the presence of phenylboronate
(Figure 4a, b). Our results indicate that the complexation
of the phenylboronate anion by the glycated peptide may
be used to distinguish glycated and non-glycated peptides.
Moreover, the higher molecular mass of phenylboronic
acid compared with the borate (Figure 4c) helps avoiding
the overlapping of signals corresponding to the resulting
complexes in complicated mixtures.
Complex Formation Using Phenylboronic Acid
QAS Derivatives
Our experiments on using borate buffer or phenylboronic
acid for selective detection of sugar-peptide conjugates gave
promising results. Therefore, we decided to further develop
this technique. We applied our strategy to the MS analysis of
trace amount of glycated peptides and non-ionizable prod-
ucts (or substances previously investigated only by negative
ion mode MS). According to the literature, one of the known
methods to increase the ionization efficiency of peptides is
their derivatization to form fixed charge ionic species[39].
The combination of an efficient method of derivatization
by phenylboronic acid and increased ionization efficiency

972
M. Kijewska et al.: Phenylboronic QAS Tags for Carbohydrates
Figure 3. ESI-MS spectrum of the Fmoc-Lys(Fru)-OH complexed with phenylboronic acid (positive ion mode) (simulated
isotopic patterns for proposed molecular formula of investigated compounds are shown)
by QAS should result in increase of cis-diols detection by
positive mode ESI-MS. We present two different strategies
of quaternary ammonium phenylboronic acid derivative
synthesis on solid support (Figures 1 and 2). In both cases,
the obtained products contain quaternary ammonium salt
and phenylboronic acid moieties. To obtain PhB-K(QAS)-
NH2 (TAG1) containing 2-(1,4-diazabicyclo[2.2.2]
octylammonium)acetyl residue as QAS, the ε-amino group
of lysine residue attached to the resin was first
iodoacetylated. Then the product was treated with an
excess of the tertiary amine. After the nucleophilic
substitution of iodine atom by a tertiary amine, the Fmoc
protecting group was removed from lysine and the
coupling of 4-carboxyphenylboronic acid was carried out
(Figure 1). The second synthetic strategy (TAG2) includes
the same steps for the formation of quaternary ammonium
salts, but the bifunctional 4-(dimethylamino)phenylboronic
acid is used for quaternary salt formation. The
iodoacetylation was performed on the free α-amino group
of glycine. Then the obtained product was treated with an
excess of the 4-(dimethylamino)phenylboronic acid. The
extension of the peptide chain (GGG) before the QAS
formation was found to improve the yield of the desired
products (Figure 2). Both applied synthetic methods
were based on the similar reactions, but they differ by
the character of amine (tertiary aliphatic in TAG1) and
substituted aniline (TAG2) as well as the order of
synthetic steps. The first method requires more steps,
but allows incorporation of more diverse QAS
moieties.
The purity and identity of the obtained tags were
confirmed by HPLC and HR-MS. The ESI-MS spectrum
of the pure PhB-K(QAS)-NH2 is presented in Supplemental
Figure S6.
Before using the synthesized tags for selective detection
of cis-diols by mass spectrometry, preliminary experiments
on ionization efficiency were conducted in water/methanol
and ammonium carbonate buffer/methanol. The application
of the same concentrations of tags (TAG1 and TAG2),
determined according to the area under the chromatogram

M. Kijewska et al.: Phenylboronic QAS Tags for Carbohydrates
973
Figure 4. (a) ESI-MS spectrum of the glycated ubiquitin hydrolysate (positive ion mode); (b) ESI-MS spectrum of the glycated
ubiquitin hydrolysate complexed with phenylboronic acid (PhB) (positive ion mode); (c) ESI-MS spectrum of the glycated
ubiquitin hydrolysate complexed with bogate (B) (positive ion mode); *glycated peptide [43-58]; **glycated peptide [43-58]
complexed with borate 644.988(3+)
signal, results in a much higher intensity of the peak
corresponding to cationic form of PhB-K(QAS)-NH2, where
the quaternary ammonium salt was formed from DABCO. In
separate experiments, the amounts of the compounds were
adjusted to generate MS peaks of comparable intensity. It
seems that the TAG2 (PhB(QAS)-GGG-NH2) is less ionized
in ESI-MS experiments than the TAG1 (PhB-K(QAS)-NH2).
It is possible that the differences arise from different charge
distribution and hydrophobic properties of these tags.
Additional experiments were performed at the same reaction
conditions as to complex formation but without addition of
carbohydrates or glycated compounds. The application of
the same concentration of tags reveals the differences in
intensity of peaks in the MS spectrum. The TAG2 shows
lower intensity in mass spectrometric experiments. All
investigated carbohydrates show comparable affinity to
phenylboronic acid derivatives used at the same concentra-
tion (Supplemental Figure S7). Therefore, further experi-
ments were performed both for the mixtures containing the
same concentration of tags and for the mixtures producing
the same intensity of their MS signals.
The lowest intensity was observed for the signals
corresponding to D-glucuronic acid, probably because
partial charge neutralization resulting from the carboxylic
group dissociation in a basic buffer. In all cases the most
abundant complex peaks correspond to the 1:1 ratio of tag–
carbohydrate concentrations. When the tag concentrations
were adjusted to give the same intensity of signals, after
incubation with carbohydrates much higher signals were
observed for TAG2 (PhB(QAS)-GGG-NH2) complexes,
especially for D-ribose and D-2-deoxyribose (Supplemental
Figure S8). It seems that the TAG2 (PhB(QAS)-GGG-NH2)
is less ionized in mass spectrometry experiments, so to
obtain the same intensity of peaks corresponding to the tags,
higher concentration of PhB(QAS)-GGG-NH2 has to be
used. The formation of the complexes with carbohydrates
changes the charge distribution in TAG2 for the benefit of
the fixed positive charge and the resulting product shows

974
M. Kijewska et al.: Phenylboronic QAS Tags for Carbohydrates
Figure 5. ESI-MS spectrum of the model peptide H-Ala-Lys(Fru)-Ala-Phe-OH complexed with TAG1: PhB-K(QAS)-NH2
(positive ion mode)
improved ionization efficiency. Therefore, the higher inten-
sities of the peaks corresponding to the complexes of sugars
with PhB(QAS)-GGG-NH2 result from the application of
larger amount of this tag to get the initial comparable signal
intensities. The proposed structures of the obtained com-
plexes for D-ribose and D-2-deoxyribose with PhB(QAS)-
GGG-NH2 and their simulated isotopic patterns are present-
ed in Supplemental Figure S9).
A series of model glycated peptides (H-Ala-Lys(Fru)-
Ala-Phe-OH, H-Asp-Thr-Glu-Lys-Gln-Ile-Lys(Fru)-Lys-
Gln-Thr-OH, H-Asp-Thr-Glu-Lys(Fru)-Gln-Ile-Lys(Fru)-
Lys-Gln-Thr-OH and the mixture of oligosaccharides [36]
were tested for their affinity towards the phenylboronic acid
derivatives.
these complexes is proven by their isotopic patterns
(Figure 5). The parent ion at m/z 504.273 was subjected to
MS/MS fragmentation using the same collision energy as in
previous experiments (Supplemental Figure S11).
The fragmentation spectrum of the model glycated
peptide labeled with PhB-K(QAS)-NH2 tag is characterized
by the low abundance of ions formed by the elimination of
water. The elimination of whole hexose moiety is also not
observed. The fragmentation spectrum is dominated by the
series of b and y ions revealing the whole sequence of
peptide. The comparison of MS/MS spectra for complexes
with borate, phenylboronate, and synthetic tags indicates that
in this case, the largest number of b and y ions was
identified.
The solution of the model glycated peptide (H-Ala-
Lys(Fru)-Ala-Phe-OH) was incubated with PhB-K(QAS)-
NH2 (TAG1) in ammonium carbonate buffer, diluted with
methanol and subjected to ESI-MS analysis. The most
abundant complex observed by MS spectra contains one
phenylboronate derivative and one molecule of the peptide;
however, a complex containing two phenylboronates was
also observed. Presumable structures for these complexes are
placed in Supplemental Figure S10). The stoichiometry of
The monoglycated and diglycated fragments of BSA (H-
Asp-Thr-Glu-Lys-Gln-Ile-Lys(Fru)-Lys-Gln-Thr-OH and H-
Asp-Thr-Glu-Lys(Fru)-Gln-Ile-Lys(Fru)-Lys-Gln-Thr-OH)
were incubated with different amounts of PhB-K(QAS)-NH2
(TAG1) in ammonium carbonate buffer, diluted with
methanol and subjected to ESI-MS analysis. The represen-
tative spectra are presented in Supplemental Figures S12 and
S13. The ESI spectra indicate the interactions of the peptide
with phenylboronate derivative. A series of complexes

M. Kijewska et al.: Phenylboronic QAS Tags for Carbohydrates
975
The formation of the phenylboronate derivatives complexes
was also studied on the oligosaccharide isolated from Hafnia
alvei PCM (Polish Collection of Microorganisms) 1200
lipopolysaccharide (LPS). H. alvei is a gram-negative bacterium
and a causative agent of respiratory diseases, mixed hospital
infections, bacteremia, and septicemia in humans [36]. The
oligosaccharide is composed of one repeating unit (RU) of the
LPS 1200 O-specific polysaccharide devoid of terminal Glc
residue (Quip4NAc + GroP + Gal + 2xGlcpNAc) linked to the
Hep-Kdo disaccharide. It is the shortest form of the O-specific
chain present on bacterial surface. Two variants were
identified—substituted with and devoid of one O-acetyl group
(Ac). Before incubation with phenyboronate derivative (TAG1 -
PhB-K(QAS)-NH2), the oligosaccharide may be analyzed only
in negative ion mode MS (Figure 6a). After the incubation, the
oligosaccharides may also be investigated in positive ion mode
MS (Figure 6b). In the spectrum, the series of complexes are
observed (Figure 6b). The isotopic patterns of these complexes
are typical for compounds containing boron atom.
Conclusions
The new tags based on the derivatives of phenylboronic acid
were designed, synthesized according to the solid phase Fmoc-
strategy, and applied for selective detection of sugars and
peptide-sugar conjugates in mass spectrometry. The formation
of complexes between sugar or sugar-peptide conjugates with
reagents (borate, phenylboronate, phenylboronate derivatives)
was confirmed on the basis of the unique isotopic distribution
resulting from the presence of boron atom. The straightforward
and convenient method based on the formation of borate and
phenylboronate complexes of carbohydrates and peptide-
derived Amadori products and their specific behavior in CID
experiments was developed. For all reagents (borate,
phenylboronate and phenylboronate derivatives), the stabiliza-
tion of hexose moiety in MS/MS was observed. The neutral
losses are significantly reduced in respect to free peptide-
derived Amadori products; therefore the fragmentation spectra
are simplified. The complexation of the phenylboronate or
borate ion by the glycated peptide may be used to distinguish
glycated and non-glycated peptides. Moreover, the higher
molecular mass of phenylboronic acid compared with the
borate limits the risk of overlapping signals corresponding to
the resulting complexes in complicated mixtures. Unfortunate-
ly, interactions of glycated peptides with QAS-containing
phenylboronate tags did not result in significant increase in
sensitivity of glycated peptides detection. On the other hand,
the ionization of monosacharides as well as natural mixture of
lipoplysacharides in positive ion mode was enhanced. The
combination of strategies involving phenylboronic acids and
QAS is a promising method for sensitive detection of
carbohydrates by electrospray ionization tandem mass spec-
trometry. Moreover, studies on sugars–phenylboronic acid
interactions may be useful for the development of carbohydrate
receptors.
Figure 6. ESI-MS spectra of the oligosaccharides isolated
from H. alvei LPS built of one RU linked to the Hep-Kdo
disaccharide: (a) sample dissolved in ammonium carbonate
buffer (negative ion mode); (b) sample with TAG1 dissolved in
ammonium carbonate (positive ion mode); (M1, M2: 3-deoxy-
D-manno-oct-2-ulosonic acid – Kdo; repeating unit – RU; L-
glycero-D-manno-heptose – Hep, Ac – O-acetyl group)
containing one phenyboronate anion are observed [597.3042
(3+) for monoglycated peptide; 651.343 (3+) for diglycated
peptide], however, a complex containing two
phenylboronate ions was also observed for peptide contain-
ing two hexose moieties (Supplemental Figure S13). The
application of increased concentrations of the tag does not
lead to complete labeling of glycated peptide contrary to
what was observed for model glycated tetrapeptide. The
repulsion of positive charges located on the peptide
molecule may be responsible for this phenomenon. The
intensity of the observed complex ion is high enough to
easily identify it in the MS spectrum.
The mixture of mono- and diglycated peptides was
incubated with the mixture of tags. The tags were used in the
amounts selected to obtain the initial MS peaks of comparable
intensity. Supplemental Figures S14 shows a series of mixed
complexes containing one or two phenylboronate derivatives.
The affinity of tags for monoglycated and diglycated peptides
is comparable because the intensity of peaks corresponding to
the complexes is similar.

976
M. Kijewska et al.: Phenylboronic QAS Tags for Carbohydrates
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This work was supported by grants 2274/M/WCH/12 at
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Ministry of Science and Higher Education of Poland, and
UMO-2012/07/D/ST5/002324 from National Science Centre
of Poland.
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