Cent. Eur. J. Chem. • 11(8) • 2013 • 1309-1319
DOI: 10.2478/s11532-013-0265-9
Central European Journal of Chemistry
High resolution mass spectrometric
characterization of amino linked
oligosaccharides - a preliminary study
Research Article
Roxana M. Ghiulai1, Mirela Galusca2,3,
Ioana Sisu1, Eugen Sisu1, Alina D. Zamfir2,4*
1
Department of Biochemistry,
University of Medicine and Pharmacy “Victor Babes”,
300041 Timisoara, Romania
2
Department of Chemistry and Biology,
“Aurel Vlaicu” University of Arad,
310130 Arad, Romania
3
Department of Physics,
West University Timisoara,
300223 Timisoara, Romania
4
Mass Spectrometry Laboratory,
National Institute for Research and Development
in Electrochemistry and Condensed Matter,
300224 Timisoara, Romania
Received 1 February 2013; Accepted 26 March 2013
Abstract: In
this study maltose, maltotriose and maltotetraose were for the first time, coupled to 4,4’-methylenedianiline (MDA). The aim of
this preliminary work was to test the feasibility of oligo- and polysaccharide coupling to MDA and the characterization of the coupling
products by high resolution mass spectrometry (MS). (+) nanoESI in combination with a quadrupole time of flight (QTOF) MS in
full scan (MS) and MS/MS was optimized first on underivatized maltose, maltotriose and maltotetraose. The optimal screening and
sequencing conditions were further applied to the MDA-functionalized oligosaccharides.The obtained results revealed a straightforward
MS detection of the functionalized oligomers, high sequence coverage and a fragmentation pathway with the formation of B and Y ions
as well as the complementary C and Z ions along with a typical cleavage of the aglycon. We consider that this methodology is fully
applicable also to polydisperse mixtures of long chain polysaccharides, which due to the large number of components and their size
require a systematic method of development and testing.
Keywords: MDA-functionalized oligosaccharides • Nanoelectrospray • QTOF MS • CID MS/MS
© Versita Sp. z o.o.
1. Introduction
Complex carbohydrate polymers covalently attached to
proteins or lipids, glycoconjugates, act as signals that
determine the intracellular location or metabolic fate of
these hybrid molecules [1].
Carbohydrates are polyhydroxy aldehydes or
ketones, or substances that yield such compounds
on hydrolysis. There are three major classes of
carbohydrates: monosaccharides, oligosaccharides,
and polysaccharides. Monosaccharides, or simple
sugars, consist of a single polyhydroxy aldehyde or
ketone unit. The most abundant monosaccharide in
nature is the six-carbon sugar D-glucose, sometimes
referred to as dextrose [1]. Oligosaccharides consist
of short chains of monosaccharide units, or residues,
joined by characteristic linkages called glycosidic bonds.
The most abundant are the disaccharides, with two
* E-mail: [email protected]
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High resolution mass spectrometric characterization
of amino linked oligosaccharides - a preliminary study
monosaccharide units. In cells, most oligosaccharides
consisting of three or more units do not occur as free
entities but are joined to non-sugar molecules (lipids or
proteins) in glycoconjugates [1].
Polysaccharides
form
homopolymers
and
heteropolymers bearing a large number of
monosaccharide units joined by glycosidic bonds. A
variety of natural polysaccharides, may be subjected
to chemical modifications for the synthesis of hybrid
materials [2,3]. Research has been undertaken to
produce block copolymers with amphiphilic properties.
Functionalization of non-polar polymer matrices with
hydrophilic carbohydrates was carried out [4] to enable
biochemical or biological reactions at the polymer
surface. Block copolymers [5,6] are soft materials formed
by two or more chemically homogeneous polymer blocks
joined together by covalent linkages. Amphiphilic block
copolymers are of high importance in medicine and
pharmacy as drug delivery systems [7-10], nanocarriers
[11], biomimetic membranes [12], surfactants for stem
cell differentiation [13] and in industry for non-biofouling
materials [14].
One of the most efficient methods for copolymer
synthesis is based on the coupling of functionalized
hydrophobic blocks to hydrophilic moieties. Such
methods can be successfully applied for the modification
of polysaccharide reducing end, as previously described
[15-17].
We report here on the first coupling of maltose,
maltotriose and maltotetraose to 4,4’-methylenedianiline
(MDA), in a preliminary study for testing the feasibility
of oligo- and polysaccharide coupling to MDA. MDA
is employed as a chemical intermediate in plastics
processing for high performance polymers, for
polyurethane elastomers, foams and special-purpose
coatings as well as for epoxy resins and two-component
systems (CAS no. 101 - 77 – 9).
Our goal was to apply our previously developed
synthesis method [15-17] for the functionalization with
MDA of oligosaccharides and to develop high resolution
mass spectrometry for the characterization of resulting
oligosaccharide derivatives. We have employed the
reductive amination reaction in order to create a new
C–N bond between the carbohydrate, which exists in
solution either in a cyclic hemiacetal or in open-chain
aldehyde form and a diamine residue.
Coupling products were characterized by positive
ion mode nanoelectrospray ionization (nanoESI)
quadrupole time-of-flight (QTOF) mass spectrometry
(MS) and tandem mass spectrometry (MS/MS) method,
particularly optimized for detection and sequencing of
MDA-linked oligosaccharides.
1310
2. Experimental procedure
2.1. Materials
Acetonitrile (99.9% purity), ethanol (99.9% purity),
acetone (99.8% purity) and DMF (99.9% purity), were
purchased from Merck (Darmstadt, Germany) and used
without further purification. Maltose (99% purity) was
purchased from Sigma (Germany), maltotriose (96%
purity) from BioChemika and maltotetraose (96% purity)
from Supleco (USA). For sample solution preparation
deionized water obtained by using a system from SG
Water (Germany) was used. All sample solutions were
dried in a SpeedVac Concentrator, SPD 111V-230
(Thermo Electron Corporation, Asheville, NC, USA)
coupled to a vacuum pump, PC 2002 Vario with CVC
2000 Controller (Vaccubrand, Wertheim, Germany).
The sample solutions were dialyzed against water
using MWCO 500 Micro DispoDialyzer from Harvard
Apparatus.
Nano ESI experiments were carried out using
self-pulled omega glass capillaries (Analytik Vertrieb,
Germany) produced on a vertical pipette puller, model
720 (David Kopf Instruments, Tujuanga, CA, USA) by an
“omega” shape filament.
2.2. Preparation of sample solutions for MS
For the synthesis of MDA-linked oligosaccharides, the
procedure previously described was applied [15-17].
For nanoESI QTOF MS experiments, the solutions
of underivatized oligosaccharides were prepared by
dissolving maltose (denoted 2G), maltotriose (3G) and
maltotetraose (4G) samples in acetonitrile:water (1:1 v/v)
to a concentration of about 10 pmol µL-1. The solution
of derivatized oligosaccharides was prepared as an
equimolar mixture of 2G_MDA, 3G_MDA and 4G_MDA
in acetonitrile:water (1:1 v/v) at the concentration of
10 pmol µL-1 for each component. Prior to MS analysis,
all sample solutions were centrifuged for 10 minutes in
a mini-centrifuge (6000 rpm) from ROTH (Germany).
The supernatants were collected and submitted to (+)
nanoESI-QTOF MS and MS/MS analysis by collision
induced dissociation (CID) at low energies.
2.3. QTOF MS and CID MS/MS
MS and CID MS/MS were performed on a hybrid
QTOF micro (Micromass/Waters, Manchester, UK)
instrument with direct nanoESI infusion in Micromass
Z-spray geometry. QTOF MS is connected to a PC
computer running the MassLynx 4.1 software to control
the instrument, acquire and process MS data. For all
acquisitions, the instrument was tuned to record the
data at a scan speed of 1 scan/s. For each screening
R. M. Ghiulai et al.
mass spectrum the signal was acquired for 5 minutes.
All mass spectra were recorded in the positive ion
mode, which was previously demonstrated to be the
best option for this type of oligosaccharide [15,16,18].
For an efficient ionization and minimal in-source
fragmentation, the cone voltage was varied within the
range of 20–50 V. The source block temperature was
set to 800C and kept at this value during all experiments.
MS/MS was performed by CID at low ion acceleration
energies using argon at 12 psi pressure as a collision
gas. For MS/MS the ions were isolated by setting the
LM and HM parameters to 10 and 10 respectively.
The product ion spectrum represents a sum of scans
combined over total ion current (TIC) scans acquired at
variable collision energy within 20-40 eV range adjusted
to provide the full set of fragment ions diagnostic for the
respective structure.
The m/z scale of the mass spectrum was calibrated
by use of an external calibration standard PEG
electrospray ‘‘tuning mix,’’ from Waters (Manchester,
UK). The reference provided in the positive ion mode
a spectrum with a high ionic coverage of the m/z range
scanned in both MS and CID MS/MS experiments. The
average mass accuracy value is situated within the
normal range of a QTOF MS instrument.
The sequence ions corresponding to the carbohydrate
moieties were assigned according to the nomenclature
introduced by Domon and Costello [19], considering
glucose-MDA as the aglycon. To designate a fragment
ion of the aglycon we have introduced the nomenclature
K and L, with K corresponding to the nonreducing end
and L to the reducing end.
3. Results and discussions
3.1. Screening and sequencing of 2G, 3G and 4G samples
The first stage for the methodological development in
analyzing the derivatized oligosaccharides was the
optimization of the system for screening and sequencing
by high resolution MS using nanoelectrospray ionization
of the precursor oligosaccharide samples used for
synthesis and derivatization.
10 µL of 2G sample solution was loaded into
the nanoelectrospray capillary and infused into the
quadrupole time-of-flight mass spectrometer. The
parameters were adjusted and optimized to minimize
the in-source fragmentation and to generate a spectrum
with high signal to noise ratio [20-22]. The screening
mass spectrum of 2G is presented in Fig. 1a.
Inspection of the spectrum indicates that the most
abundant ion was detected at m/z 365.02 assigned,
according to the mass calculation, to the monosodiated
form of 2G. Besides [2G+Na+]+ also a [2G+K+]+ was
detected at m/z 381.83. Due to the high sensitivity of
the method, at a rather low concentration of 10pmol µL-1,
up to hexamers were detected as singly charged ions at
m/z 707.09 ([2(2G)+Na+]+ ), m/z 723.10 ([2(2G)+K+]+)
and m/z 1049.20 ([3(2G)+Na+]+) or doubly charged
ions at m/z 362.03 ([2(2G)+H++K+]2+), m/z 525.07
([3(2G)+H++Na+]2+), m/z 533.05 [3(2G)+H++K+]2+),
m/z
696.10
([4(2G)+H++Na+]2+),
m/z
704.08
([4(2G)+H++K+]2+), m/z 875.15 ([5(2G)+H++K+]2+) and
m/z 1046.18 ([6(2G)+H++K+]2+). The maximum degree
of polymerization (DP) detected by (+) nanoESI QTOF
MS analysis of this mixture is six. The oligomerization of
these small molecules is a typical phenomenon occurring
in solution [23]; the high sensitivity of the method allowed
for the detection of all these oligomers even though the
concentration was at a rather low value of 10pmol µL-1.
The method was optimized for this concentration as this
value was found to be the ideal compromise between
an optimal ionization, a fair intensity of the precursor
ions necessary for CID MS/MS and the oligomerization
process.
To test and optimize the CID method for
sequencing, the ion at m/z 385.12 corresponding to the
monosodiated form of 2G, was submitted to detailed
structural investigation by fragmentation in CID MS/MS.
The spectrum obtained under variable collision energy
is depicted in Fig. 1b. A number of sodiated sequence
ions such as Y1 (or C1 because of the symmetry of
the molecule) at m/z 203.07, B1 (or Z1) at m/z 185.06
and a ring cleavage ion at m/z 305.10 corresponding
to [0,2A2+Na+]+ are diagnostic for the molecular
structure. From this spectrum we can evaluate that
the conditions for ionization and sequencing were
ideal to generate the structural data for this type of
substrates.
Maltotriose (3G) was submitted to screening under
identical conditions. The spectrum is presented in
Fig. 2a. Assessment of the spectrum indicates also
the formation of the monosodiated ion at m/z 527.09
and monopotasiated ion at m/z 543.08. Similarly to
the previous case, a number of oligomers, which
substantiate the high sensitivity of the method, were
detected. The ion at m/z 527.04 used as a precursor
to test the feasibility of the method and to establish the
sequencing conditions for 3G, was submitted to CID
MS/MS under variable conditions: cone voltage within
40-50 V, capillary voltage 2 kV and collision energy
within 20-35 eV.
The obtained spectrum is depicted in Fig. 2b. The
whole series of sodiated B/Z (B1 /Z1 at m/z 185.02; B2 /Z2
at m/z 347.03) and Y/C (Y1 /C1 at m/z 203.02; Y2 /C2 at m/z
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High resolution mass spectrometric characterization
of amino linked oligosaccharides - a preliminary study
(a)
(b)
Figure 1. Positive nanoESI QTOF MS (a) and CID MS/MS (b) of maltose.
1312
Cone voltage: 40 V. Capillary voltage: 2 kV. Acquisition: 300 scans. CID at variable collision energy within 20-35 eV. Argon pressure: 12 p.s.i.
R. M. Ghiulai et al.
(a)
(b)
Figure 2. Positive nanoESI QTOF MS (a) and CID MS/MS (b) of maltotriose sample. Cone voltage within: 40-50 V. Capillary voltage within: 2 kV. Acquisition 300 scans. CID at variable collision energy within 20-35 eV. Argon pressure: 12 p.s.i.
1313
High resolution mass spectrometric characterization
of amino linked oligosaccharides - a preliminary study
365.04) fragment ions, typical for oligosaccharides, were
generated under these conditions. These sequence ions
are accompanied by a ring cleavage ion corresponding
to [0,2A3+Na+]+ at m/z 467.05; altogether, the product ions
document the 3G molecular configuration.
The extended chain of maltotetraose was analyzed
under similar conditions. The nanoESI QTOF mass
spectrum is shown in Fig. 3a. It is obvious that in this
case, the chain of molecule being longer meant that
the oligomerization was found to be reduced to some
extent. The most abundant ions at m/z 689.07 and m/z
705.03 represent the sodiated and respectively the
potasiated forms of 4G. The ions corresponding to the
oligomerization i.e the [3(4G)+H++K+]2+ trimer ion at m/z
1019.63 and the [2(4G)+Na+]+ at m/z 1355.02 are of
lower intensity, still detected by the high sensitivity of
the nanoelectrospray ionization.
The ion at m/z 689.09 was isolated within an
isolation window with LM 10 and HM 10 and submitted
for detailed structural investigation by CID MS/MS. The
spectrum is presented in Fig. 3b. The conditions chosen
for sequencing enabled the generation of the entire
series of B/Z (B1 /Z1 at m/z 185.03; B2 /Z2 at m/z 347.05;
B3 /Z3 at m/z 509.06) together with the counterpart Y/C
series (Y1 /C1 at m/z 203.03; Y2 /C2 at m/z 365.06; Y3 /
C3 at m/z 527.09) identified as signals of fair intensity.
Besides these ions, a number of ring cleavage ions such
as [0,2A2+Na+]+ at m/z 305.07, [0,2A3+Na+]+ at m/z 467.08
and [0,2A4+Na+]+ at m/z 629.13 support the 4G structure
indicating that the sequencing conditions were properly
optimized for oligosaccharide analysis.
3.2. Screening and sequencing of 2G_MDA, 3G_MDA and 4G_MDA samples
The conditions optimized for the analysis of 2G, 3G, 4G
samples were applied for the functionalized 2G_MDA,
3G_MDA, 4G_MDA samples. This work is a preliminary
study for MDA-functionalization and MS analysis at a
large scale of polydisperse polysaccharide mixtures (i.e.,
polydisperse maltodextrins and dextran). Therefore,
to test the feasibility of the method, the ionization
conditions of these molecules in a polydisperse mixture,
the occurrence of possible ion suppression effect and
eliminate these drawbacks, we have chosen to analyze
2G_MDA, 3G_MDA, 4G_MDA samples in an equimolar
mixture.
In Fig. 4 the presence of the monosodiated ions of
2G_MDA, 3G_MDA, 4G_MDA at m/z 547.17, 709.21
and 871.33 respectively is observed. Inspecting
the spectrum, it is clear that the ion distribution
resembles the envelope shape which is characteristic
for polydisperse mixtures [24]. Another feature of the
1314
spectrum in Fig. 4 is the incidence of non-covalent
associations, a phenomenon to be taken into account
in further studies on polydisperse mixtures of average
to high molecular weight. Thus, the ion at m/z 1197.26
assigned, according to the mass calculation to [2G_MDA
+ 3G_MDA-2H2O+Na+]+ occurred as a non-covalent
association between 2G_MDA and 3G_MDA. Following
the same pattern, the non-covalent association between
2 molecules of 2G_MDA corresponds to [2x2G_MDA2H2O+Na+]+ ion observable at m/z 1035.34. The ion
at m/z 1359.47 represents a non-covalent association
between 2 molecules of 3G_MDA corresponding to
[2x3G_MDA-2H2O+Na+]+ or an association between
one molecule of 2G_MDA and 4G_MDA generating
[2G_MDA+4G_MDA -2H2O+Na+]+ ion. Also, the ions at
m/z 575.18 and 737.25 exhibit a mass value shifted with
28 Da as compared to the mass of [2G_MDA+Na+]+ and
[3G_MDA+Na+]+ respectively. This phenomenon is most
likely due to the addition of a formyl group at the terminal
amino sequence during sample preparation.
To test the feasibility of the method to isolate and
sequence the ions corresponding to 2G_MDA, 3G_MDA,
and 4G_MDA from the mixture, the first ion of the series
at m/z 547.17 corresponding to [2G_MDA+Na+]+ was
isolated and submitted to CID MS/MS. As the previous
studies have shown, [18,21,22] the spectrum presented
in Fig. 5 reveals typically B ([B1+Na+]+ at m/z 185.03) and
Y( [Y0+Na+]+ at m/z 385.14) ions. Their complementary
ions at m/z 203.05 and 367.15 corresponding to
[C1+Na+]+ and [Z0+Na+]+ respectively were also observed.
An interesting cleavage ion within the aglycon, denoted
as L, was identified as a dehydrated sodiated form of
fair intensity at m/z 187.05. A number of ring cleavage
ions at m/z 305.09, 427.19 and 457.19 corresponding
to [0,2A2+Na+]+, [0,2X+Na+]+ and [1,4X+Na+]+ respectively
were also observed, supporting the structure of 2G_
MDA.
The next ion corresponding to 3G_MDA detected in
the equimolar mixture at m/z 709.21 (Fig. 4) was isolated
and submitted to CID MS/MS (Fig. 6). According to
the spectrum presented in Fig. 6, under the employed
fragmentation conditions, 3G_MDA followed the same
fragmentation pattern as described above. The spectrum
discloses, the formation at a fair intensity of the whole
series of typical B ([B1+Na+]+ at m/z 185.04, [B2+Na+]+
at m/z 347.10) and Y ([Y0+Na+]+ at m/z 385.16 and
[Y1+Na+]+ at m/z 547.23) ions. Also their complementary
C ([C1+Na+]+ at m/z 203.05 and [C2+Na+]+ at m/z 365.09)
and Z ([Z0-H2O+Na+]+ at m/z 349.10 and [Z1+Na+]+ at m/z
529.25) ions can be observed along with several ring
cleavage ions at m/z 259.10, 305.11, 427.20 and 457.24
can be assigned, according to the mass calculation to
R. M. Ghiulai et al.
(a)
(b)
Figure 3. Positive nanoESI QTOF MS (a) and CID MS/MS (b) of maltotetraose. Cone voltage within 40-50 V. Capillary voltage: 2kV. Acquisition 300 scans. CID at variable collision energy within 20-35 eV. Argon pressure:12 p.s.i.
1315
High resolution mass spectrometric characterization
of amino linked oligosaccharides - a preliminary study
Figure 4. Positive nanoESI QTOF MS of an equimolar mixture of 2G_MDA,
2 kV. Acquisition 300 scans.
3G_MDA, 4G_MDA . Cone voltage within 20-50 V. Capillary voltage: Figure 5. Positive nanoESI QTOF CID MS/MS of 2G_MDA. Acquisition 300 scans. CID at variable collision energy within 20-40 eV. Argon pressure: 1316
12 p.s.i.
R. M. Ghiulai et al.
Figure 6. Positive nanoESI QTOF CID MS/MS of 3G_MDA. Acquisition 300 scans. CID at variable collision energy within 20-40 eV. Argon pressure: 12 p.s.i.
Figure 7. Positive nanoESI QTOF CID MS/MS of 4G_MDA. Acquisition 300 scans. CID at variable collision energy within 20-40 eV. Argon pressure: 12 p.s.i.
1317
High resolution mass spectrometric characterization
of amino linked oligosaccharides - a preliminary study
[3,5A2+Na+]+, [0,2A2+Na+]+, [0,2X1+Na+]+ and [1,4X1+Na+]+
respectively. Moreover, the aglycon cleavage ion at m/z
205.12 corresponding to [L+Na+]+ was again detected.
Finally, the ion at m/z 871.30 corresponding to
[4G_MDA +Na+]+ from the equimolar mixture was also
isolated and submitted to CID MS/MS. Analysis of
the spectrum in Fig. 7 indicates that under carefully
optimized fragmentation conditions in terms of collision
energy and gas pressure values, the generated product
ions cover the entire sequence of 4G_MDA compound.
The ions diagnostic for the 4G_MDA structure are:
i) the whole series of typical B ions, [B1+Na+]+ at m/z
185.07, [B2+Na+]+ at m/z 347.14 and [B3+Na+]+ at m/z
509.16 and Y ions, [Y0+Na+]+ at m/z 385.23, [Y1+Na+]+
at m/z 547.28 and [Y2+Na+]+ at m/z 709.32; ii) the
complementary [C2+Na+]+ at m/z 365.11 and the Z
series including [Z0+Na+]+ at m/z 367.17 and [Z1+Na+]+
at m/z 529.24 ions; iii) a ring cleavage ion at m/z 305.13
corresponding to [0,2A2+Na+]+; iv) the aglycon-related
cleavage ion at m/z 205.09 corresponding to [L+Na+]+.
Comparative assessment of these results reveals
that obviously there is a similar fragmentation pathway of
2G_MDA, 3G_MDA and 4G_MDA with the formation of
B and Y ions as previously reported and the occurrence
of complementary C and Z ions along with the typical
cleavage of the aglycon. These aspects require a
particular consideration when such amino-derivatized
oligosaccharides are submitted to CID MS/MS.
4. Conclusions
In this study we have achieved for the first time the
synthesis and structural characterization of amino linked
oligosaccharides: 2G_MDA, 3G_MDA and 4G_MDA. As
a preliminary study for implementing the method at a
large scale for the analysis of polydisperse mixtures of
long chain polysaccharides, the goal of this work was
to determine the optimal ionization and sequencing
conditions of this class of derivatized carbohydrates, their
behavior in polydisperse mixtures and their fragmentation
pattern under identical CID conditions. The optimized
detection and fragmentation conditions have led to a
straightforward molecule identification. Therefore, we
consider that the method can be successfully applied
to polydisperse mixtures of long chain polysaccharides,
which due to the large number of components and their
size, demand as the analysis prerequisite, a thorough
method of development and testing.
Acknowledgements
This work was supported by the European Social Fund,
through the project POSDRU 107/1.5/S/78702 and by
the Romanian National Authority for Scientific Research
through the projects PN-II-ID-PCE-2011-3-0047 and
PN-II-PCCA-2011-142.
Abbreviations
MDA: 4,4’-Methylenedianiline;
G: glucose;
2G: maltose;
3G: maltotriose;
4G: maltotetraose;
2G_MDA: MDA-linked maltose;
3G_MDA: MDA-linked maltotriose;
4G_MDA: MDA-linked maltotetraose;
CID: collision-induced dissociation;
ESI: electrospray ionization;
MS/MS: tandem mass spectrometry;
TIC: total ion chromatogram;
QTOF MS: quadrupole time-of-flight mass spectrometer/spectrometry;
PEG: polyethyleneglycol;
1318
R. M. Ghiulai et al.
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