EXPERIMENTAL

1
Characterization of Oligosaccharides Using
Infrared Multi-Photon Dissociation and Chemical
Derivatization in a Quadrupole Ion Trap Michael
Pikulski, Lisa Vasicek, Amanda Hargrove, Shagufta
H. Shabbir, Eric Anslyn, Jennifer S. Brodbelt
Department of Chemistry and Biochemistry The
University of Texas at Austin
LNDFH Oligosaccharides This characterization
strategy was expanded to the second
oligosaccharide series and the same two pathways
of dissociation that were observed for the LNFP
series were seen again (Figure 7). The primary
fragmentation pathway provides detailed sequence
information for the LNDFH oligosaccharides with
the exception of the two isomers that only differ
in their reversed Gal and Fuc linkages to the
central GlcNAc (Ia and Ib). Following the loss
of terminal or branching Fuc moieties,
fragmentation of the backbone for each of the
isomers from the non-reducing end starts with the
combined Gal-GlcNAc losses followed by two
sequential Hex losses (Gal and Glc). Other
possible starting sequences of these
oligosaccharides are discounted because in each
of the IRMPD spectra throughout this study there
has been a key intersaccharide cleavage at the
reducing end of GlcNAc. These fragments ions are
not observed in the IRMPD mass spectra, thus
discrediting these alternative sequences.
Therefore the two Fuc must reside on the
non-reducing end Gal and GlcNAc. The difference
in the IRMPD spectrum for the LNDFH-II isomer is
a result of a Fuc having a different position.
Since the initial Fuc loss precedes the
Gal-GlcNAc combined loss, it is evident that this
Fuc resides on either the Gal or GlcNAc

• OVERVIEW
• A derivatization agent with high IR absorptivity,
  IRABA, was designed and synthesized to enhance
  the reactivity of the boronic acid functionality
  with oligosaccharides and improve the ability to
  sequence oligosaccharides without the need for
  MSn.
• Two series of oligosaccharides were studied the
  lacto-N-fucopentaoses (LNFPs), and the
  lacto-N-difucohexaoses (LNDFHs).
• The IRABA-oligosaccharide products dissociated
  with high efficiency upon IR irradiation.
• The resulting IRMPD mass spectra display a
  characteristic primary fragmentation pathway that
  simplifies the determination of oligosaccharide
  sequence.

EXPERIMENTAL Derivatization The IRABA group was
synthesized by a modification to a synthesis
described by the Ansyln group (College of
Chemistry and Biochemistry, University of Texas
at Austin)3. First, 3.9 mmol 2-formylphenylboroni
c acid (mM) was dissolved in anhydrous CH3OH
under argon protection. Then 16 mmol Hunigs base
was added followed by 3.9 mmol diethyl(aminomethyl
)phosphonate oxalate. The solution was stirred
for 16 hours before 2.9 mmol NaBH4 was added
slowly, stirred an additional hour, followed by
another addition of NaBH4. One hr later, the
solvent was removed under vacuum and the residue
was diluted with CH2Cl2. The white ppt was
removed with vacuum filtration, with the filtrate
subsequently concentrated. The residue was
purified by flash chromatography on neutral
alumina (2-5 NH3-saturated CH3OH in CH2Cl2) and
at a final concentration of10 µM mixed with stock
solutions of the glycans (500 µM) to make a 110
molar ratio along with a 5 µL aliquot of 0.5 TEA
was added to a pH 9. The solutions were then
sonicated for 1 min and diluted with 0.1 TEA to
pH 8 and spiked with ammonium acetate (to
0.01). Mass Spectrometry A 10 µM
oligosaccharide solution was injected into a
Finnigan LCQ Deca XP mass spectrometer equipped
with an electrospray ionization source and
interfaced with a Synrad CO2 50 W laser (10.6
µm). Typical IRMPD parameters included an
irradiation time of 5 - 25 msec at a power of 50
W and a helium pressure of 2.8 x 10-5 Torr. For
the underivatized oligosaccharides, the helium
pressure was lowered to nominally 2.7 x 10-5 Torr
to promote the IRMPD process.
INTRODUCTION Oligosaccharides are in many ways
more structurally complex than other biologically
important molecules such as proteins and nucleic
acids in that they are often highly branched and
have several different linkage types between
their fundamental monosaccharide units. The
complex structures of oligosaccharides result in
their characterization a challenging but vital
task because they have important roles in
numerous biological processes. Mass
spectrometry (MS) has become an invaluable tool
for the structural determination of glycans
mainly due to the minimal sample consumption and
specificity of the information obtained. To
provide more extensive structural information and
alleviate the need for elaborate MSn strategies,
IRMPD1 has been implemented in quadrupole ion
traps and affords a promising alternative to CAD
for the characterization of oligosaccharides. In
the present study, we report a simplified method
for the sequencing of oligosaccharides in a
single stage of activation by exploiting the
supplementary information obtained from
sequential fragmentation that is promoted by
non-resonance IRMPD and derivatization with a
boronic acid that has an incorporated phosphonate
group, IRABA, to increase IR absorption
efficiency.
The first general type of pathway is defined by
sequential losses from the non-reducing end of
the oligosaccharides (losses highlighted in black
type) and correspond to a loss of Fuc and a
combined loss of Hex and HexNAc for the LNFPs.
The persistent combined loss of Hex and HexNAc
suggests that cleavage at the non-reducing end of
the GlcNAc is not a facile process. The second
general pathway observed in Figure 5 involves the
combined loss of two Hex and W arising from
cleavage at the reducing end of the
oligosaccharide (losses highlighted in blue
type). However, in this CAD spectra the sequence
coverage of the oligosaccharides is still
limited, therefore IRMPD was used to further the
study.
LNDFHIaIRABA-2H2OH
IRABA Derivatized Oligosaccharides Since neither
the CAD nor the IRMPD spectra of the
underivatized LNFPs yielded diagnostic fragment
ions or sufficient sequencing information for the
isomers, the boronic acid derivatization strategy
was explored next.
-ESI ESI
RESULTS AND DISCUSSION LFNP Oligosaccharides CAD
and IRMPD spectra of both the underivatized and
derivatized species were taken to develop a
method for sequencing the backbone of the LNFPs
and to pinpoint the sites of attachment of the
fucose moieties. The CAD mass spectra of the
deprotonated oligosaccharides, (L - H-), are
shown in Figures 2a-d.
Figure 1 A scheme showing the derivatization
strategy for oligosaccharides.
LNFPIIRABA-2H2ONa
The IR-active boronic acid (IRABA) was designed
so that a nitrogen atom was adjacent to the boron
atom to enhance the reactivity of the boronic
acid functionality to the oligosaccharides2.
When the IRABA is added to a solution containing
an oligosaccharide of interest, the chemical
reaction between the IRABA and diol
functionalities of the oligosaccharide is
responsible for the covalent addition of the
IR-active phosphonate group. Two series of
oligosaccharides were studied, the
lacto-N-fucopentaoses (LNFPs), all have the same
backbone sequence but differ either in the
connectivity of fucose (Fuc) or the linkage of
their terminal Gal and Fuc moieties, and the
lacto-N-difucohexaoses (LNDFHs), also have the
same backbone structure but have two Fuc residues
and differ either in the connectivity or linkage
of one of the Fuc residues. The IRABA-modified
analytes proved to be ideal for characterization
and differentiation using IRMPD.
Structures of Oligosaccharides
m/z
Figure 4 ESI mass spectra for IRABA derivatized
LNFP-I in both the negative and positive ion
modes. The IRABA-derivatization efficiency is
very high.
Figure 7 IRMPD spectra of the IRABA derivatized
LNDFH oligosaccharides
LNFP
LNDFH
In negative mode, there is no indication of
whether the Hex residues are cleaved from the
reducing or non-reducing ends. Therefore, the
analytical value is similar to that obtained from
the CAD and IRMPD spectra of the deprotonated
underivatized oligosaccharides. However in
positive mode, the data suggests that there are
two general dissociation pathways the most
prominent one from sequential losses from the
non-reducing end of the oligosaccharides (a
result of Y-type cleavages) and the other
entailing losses from the reducing end (a result
of B-type cleavages).
DSLNT Oligosaccharide The IRABA derivatization
strategy was further extended to a hexasaccharide
dicarboxylic acid, disialyllacto-N-tetraose(DSLNT)
. As observed with the previous series, the
principal fragments are a series that sequence
the backbone from the non-reducing to reducing
end of the oligosaccharide (Figure 8). Following
the loss of the two Sia moieties, a combined loss
of Gal-GlcNAc resulting from cleavage at the
reducing end of GlcNAc indicates the sequence
Sia-Gal-(Sia)-GlcNAc. Subsequent losses reveal
the completion of the sequence, Gal-Glc. The
successful sequencing of this oligosaccharide
suggests that the method may be further applied
to larger and different types of
oligosaccharides.
LNDFH-I(a) Fuca1-2Galß1-3(Fuca1-4)GlcNAcß1-3Galß1
-4Glc
DSLNTIRABA-2WH
Y4a
LNDFH-I(b) Fuca1-2Galß1-4(Fuca1-3)GlcNAcß1-3Galß1-
4Glc
(-Fuc)
Figure 6 IRMPD spectra for the IRABA derivatized
LNFPs
In addition to the same losses observed in the
CAD spectra, the IRMPD spectra also reveal ions
that have undergone subsequent dissociation.
These secondary IRMPD events are responsible for
the two additional Hex losses observed for all of
the LNFPs in Figures 6. As a result, there is
complete sequence coverage for each of the LNFPs.
In addition, the diagnostic pathway for the
LNFP-V is present (initial loss of Gal GlcNAc)
along with a diagnostic ion for the LNFP-I isomer
that was not observed in the CAD spectra (Y2).
It is surmised that this ion is due to sequential
fragmentation of the fragment ion at 973 Da due
to the initial loss of Fuc. We believe the
differences in location of the charge site after
the initial loss of Fuc for LNFP-I with a
non-reducing terminal Fuc, LNFP-II and III  with
a GlcNAc bound Fuc, and LNFP-V with a Glc bound
Fuc are what make this loss for the LNFP-I isomer
possible. The primary fragmentation pathway is
the key to reliable sequencing of the
oligosaccharides since the fragments that occur
in this pathway only occur from one end of the
oligosaccharide. This primary fragmentation
pathway is observed only when the
oligosaccharides are derivatized. Therfore,
derivatization coupled with IRMPD has proven to
be an excellent technique for sequencing the
LNFPs in one activation event. Site of
Derivatization The losses observed in the primary
fragmentation pathway in the IRMPD spectra
suggest that the IRABA is attached to the
reducing sugar. Since the reducing sugar is the
most reactive and may have both the alpha and
beta configuration, it may bear the cis-diol
functionality that readily reacts with boronic
acids. The existence of the secondary pathway
suggests that there is at least one other site of
derivatization. Since the first losses in the
secondary pathway include two Hex and Fuc, it is
surmised that the secondary site of attachment is
at one of the Gal moieties that also possess the
cis-diofunctionality. To further elucidate the
site of attachment, the reducing end of the
LNFP-II isomer was reduced to an alditol with
sodium borohydride. The alditol form of LNFP-II
was then reacted with the IRABA ligand, followed
by analysis by ESI-MS. No product of
derivatization was observed in the spectra.
Given these results, it is surmised that the
IRABA reacts primarily at the reducing sugar and
that any other sites of derivatization are minor
products.
LNDFH-II Galß1-3(Fuca1-4)GlcNAcß1-3Galß1-4(Fuca1-
3)Glc
CONCLUSIONS The sequencing of oligosaccharides
has been simplified with the use of IRMPD in a
QIT. This was accomplished with the use of a
boronic acid derivatizing reagent that was also
functionalized with an IR-active phosphonate
group to facilitate the photon absorption
process. The oligosaccahrides underwent
modification by simple addition of the IRABA with
reaction times of 1 min and did not require
sample cleanup prior to analysis by ESI-MS. The
resulting IRABA-oligosaccharide products
dissociated with high efficiency upon IR
irradiation, and the degree of secondary
fragmentation may be controlled by adjusting the
irradiation time. The resulting IRMPD mass
spectra display a characteristic primary
fragmentation pathway that consisted of one
uniform type of ion, thereby simplifying the
determination of oligosaccharide sequence. As a
result, the method should be generally applicable
to the sequencing of even larger unknown
oligosaccharides.
Figure 8 IRMPD of IRABA derivatized
negatively-charged hexasacharide,
disiayllacto-N-tetraose (DSLNT)
LNFP-V Galß1-3GlcNAcß1-3Galß1-4(Fuca1-3)Glc
m/z
Figure 3 CAD and IRMPD spectra of the
deprotonated oligosaccharides
The spectra for the first three isomers (Figure
3a-c) all result in losses of a hexose residue
(Hex, -162 Da) as a Y- or B-type cleavage,
therefore not providing any valuable structural
information. In the CAD spectrum for LNFP-V
(Figure 3d), a loss of 164 Da indicates the
elimination of a fucose residue (Fuc) with water
(H2O). This additional water loss is indicative
of a Z- or C-type cleavage where the
intersaccharide oxygen atom is associated with
the non-reducing end of the oligosaccharide upon
cleavage. In addition, a loss of two saccharide
groups, both hexose and fucose residues, is
observed. The IRMPD spectra with an increase in
the laser irradiation time resulted in an
increase in intensity of lower mass fragments
coupled with the decrease in intensity of higher
mass fragments. The spectrum for deprotonated
LNFP-I through LNFP-V are displayed above (Figure
3e-h). Unfortunately, for each isomer there is a
lack of a significant amount of information for
sequencing the backbone and Fuc position. In
addition, the sequential losses are limited to
one to three monosaccharide units, thus
restricting the coverage of information obtained.
Due to the unsatisfactory structural
characterization of the deprotonated
oligosaccharides, the CAD and IRMPD spectra of
the sodium-cationized compounds were obtained for
comparison. In the positive ion mode,
sodium-cationized complexes were formed almost
exclusively over the protonated species, and thus
they were therefore used for the analyses.
ACKNOWLEDGEMENTS Support from the Robert A. Welch
Foundation (F1155) and the National Science
Foundation (CHE-0315337) is gratefully
acknowledged.
REFERENCES (1) Wilson, J. J. Brodbelt, J.S. A.
Chem. 2006, 78, 6855-6862. (2) Zhu, L. Shabbir,
S. H. Gray, M. Lynch, V. Sorey, S. Anslyn, E.
V. J. Am. Chem. Soc. 2006, 128, 1222-1232.
(3) Zhu, L. Anslyn, E. V. J. Am. Chem. Soc.
2004, 126, 3676-3677.
Glc
Fuc
Gal
GlcNAc
Sia
Figure 5 CAD spectra for the derivatized LNFPs
of the type LNFPIRABA-2H2OH
Figure 2 Structures of LNFP and LNDFH
oligosaccharides