Epitope Ligand Binding Sites of Blood Group Oligosaccharides in Lectins Revealed by Pressure-Assisted Proteolytic Excision Affinity Mass Spectrometry

Article abstract of DOI:10.1007/s13361-018-1998-7

Affinity mass spectrometry using selective proteolytic excision and extraction combined with MALDI and ESI mass spectrometry has been applied to the identification of epitope binding sites of lactose, GalNac, and blood group oligosaccharides in two blood group-specific lectins, human galectin-3 and glycine max lectin. The epitope peptides identified comprise all essential amino acids involved in carbohydrate recognition, in complete agreement with available X-ray structures. Tryptic and chymotryptic digestion of lectins for proteolytic extraction/excision-MS was substantially improved by pressure-enhanced digestion using an automated Barocycler procedure (40?kpsi). Both previously established immobilization on affinity microcolumns using divinyl sulfone and coupling of a specific peptide glycoprobe to the gold surface of a biosensor chip were successfully employed for proteolytic excision and extraction of carbohydrate epitopes and affinity measurements. The identified epitope peptides could be differentiated according to the carbohydrate employed, thus demonstrating the specificity of the mass spectrometric approach. The specificities of the epitope ligands for individual carbohydrates were further ascertained by affinity studies using synthetic peptide ligands with immobilized carbohydrates. Binding affinities of the synthetic ligand peptides to lactose, in comparison to the intact full-length lectins, were determined by surface acoustic wave (SAW) biosensor analysis and provided micromolar K_D values for the intact lectins, in agreement with results of previous ITC and SPR studies. Binding affinities of the epitope peptides were approximately two orders of magnitude lower, consistent with their smaller size and assembled arrangement in the carbohydrate recognition domains. [Figure not available: see fulltext.].

Full text of DOI:10.1007/s13361-018-1998-7

B American Society for Mass Spectrometry, 2018
J. Am. Soc. Mass Spectrom. (2018)
DOI: 10.1007/s13361-018-1998-7
RESEARCH ARTICLE
Epitope Ligand Binding Sites of Blood Group
Oligosaccharides in Lectins Revealed by Pressure-Assisted
Proteolytic Excision Affinity Mass Spectrometry
Yannick Baschung,1,2 Loredana Lupu,1 Adrian Moise,3 Michael Glocker,2 Stephan Rawer,4
Alexander Lazarev,5 Michael Przybylski1,3
1Steinbeis Centre for Biopolymer Analysis and Biomedical Mass Spectrometry, Marktstraße, 29, 65428, Rüsselsheim am Main,
Germany
2Department of Immunology, University of Rostock, Rostock, Germany
3Department of Chemistry and Steinbeis Center for Biopolymer Analysis and Biomedical Mass Spectrometry, University of
Konstanz, 78457, Konstanz, Germany
4Thermofisher Scientific, Frankfurter Straße 134, Darmstadt, Germany
5Pressure BioSciences, Inc., 14 Norfolk Ave., South Easton, MA, USA
Abstract. Affinity mass spectrometry using selec-
tive proteolytic excision and extraction combined
with MALDI and ESI mass spectrometry has been
applied to the identification of epitope binding sites
of lactose, GalNac, and blood group oligosaccha-
rides in two blood group-specific lectins, human
galectin-3 and glycine max lectin. The epitope
peptides identified comprise all essential amino
acids involved in carbohydrate recognition, in
complete agreement with available X-ray struc-
tures. Tryptic and chymotryptic digestion of lectins for proteolytic extraction/excision-MS was substantially im-
proved by pressure-enhanced digestion using an automated Barocycler procedure (40 kpsi). Both previously
established immobilization on affinity microcolumns using divinyl sulfone and coupling of a specific peptide
glycoprobe to the gold surface of a biosensor chip were successfully employed for proteolytic excision and
extraction of carbohydrate epitopes and affinity measurements. The identified epitope peptides could be differen-
tiated according to the carbohydrate employed, thus demonstrating the specificity of the mass spectrometric
approach. The specificities of the epitope ligands for individual carbohydrates were further ascertained by affinity
studies using synthetic peptide ligands with immobilized carbohydrates. Binding affinities of the synthetic ligand
peptides to lactose, in comparison to the intact full-length lectins, were determined by surface acoustic wave (SAW)
biosensor analysis and provided micromolar KD values for the intact lectins, in agreement with results of previous
ITC and SPR studies. Binding affinities of the epitope peptides were approximately two orders of magnitude lower,
consistent with their smaller size and assembled arrangement in the carbohydrate recognition domains.
Keywords: Mass spectrometry, Human galectin-3, Glycine max lectin, Blood group oligosaccharides, CRD,
Recognition sites, Proteolytic excision, proteolytic extraction, Ligand epitope peptides, SAW-biosensor analysis
AbbreviationsCRDCarbohydrate recognition domain; AoaAminooxyacetic acid; DVSDivinyl sulfone;
GalNAcAcétylgalactosamine; PBSPhosphate-buffered saline; DTTDithiothreitol; SAMSelf-assembled monolay-
er; SAWSurface acoustic waves
Received: 16 March 2018/Revised: 15 May 2018/Accepted: 22 May 2018
Electronic supplementary material The online version of this article (https://
doi.org/10.1007/s13361-018-1998-7) contains supplementary material, which
is available to authorized users.
Correspondence to: Michael Przybylski; e-mail: Michael.Przybylski@stw.de

Y. Baschung et al.: Oligosaccharide Epitopes in Lectins by Affinity-MS
Introduction
Experimental
nteractions of carbohydrates with proteins such as lectins
belong to the most important types of biological process-
Lectins and Proteolytic Enzymes
I
Recombinant human galectin-3 was obtained from
Carbosynth, Compton (UK). Hgal-3 was purified from extracts
of bacteria by affinity chromatography on lactosylated Sepha-
rose 4B and analyzed for purity by one- and two-dimensional
gel electrophoresis and gel filtration as previously described [2,
3]. It was checked for activity by solid-phase and cell-based
assays [3, 8]. Glycine max lectin isolated from soy bean was
purchased from Medicago AB (Uppsala, Sweden) and was
purified by affinity chromatography. Trypsin and chymotryp-
sin, sequencing grade, were obtained from Promega (Mann-
heim, Germany).
es, e.g., in cellular recognition, intracellular regulation path-
ways, and immunological reactions. Complete structures of
oligo- and polysaccharide complexes with lectins and anti-
bodies have been determined in a few cases both by X-ray
crystallography and by nuclear magnetic resonance spec-
troscopy. Crystal structures of complexes with glycans have
been defined for antibodies, plant and animal lectins, bac-
terial toxins, and carbohydrate-binding enzymes. However,
x-ray crystallography approaches are limited by the notori-
ous difficulties in crystallization of protein-glycan com-
plexes at sufficiently high resolution, as well as by the high
requirements of relatively large amounts and highly purified
samples [1].
Carbohydrate Ligands
We have recently developed a new approach for the
molecular identification of carbohydrate recognition epi-
topes in lectins with high specificity, sensitivity, and low
requirements of sample purity [2, 3], using a combination
of proteolytic digestion and mass spectrometry. In a first
variant of the analytical approach termed proteolytic exci-
sion [4–7], the carbohydrate is immobilized on an affinity
column and the carbohydrate-binding protein (lectin, anti-
body, etc.) added to form a complex which is then sub-
jected to proteolytic digestion. In a second variant, proteo-
lytic extraction, the protein is first subjected to proteolytic
digestion and the mixture of peptide fragments presented
to the immobilized carbohydrate. After washing away the
non-binding peptides, the remaining affinity-bound peptide
ligand fragments are eluted and analyzed by mass spec-
trometry (Fig. 1a). This approach has been generally
termed BCarbohydrate Recognition Domain Excision
(BCREDEX^) [3].
In the CREDEX approach, the proteolytic step has been
often a major problem, since intact lectins are highly stable
to enzymatic hydrolysis and require time-consuming proteo-
lytic digestion. Here, we demonstrate the application of
high-pressure proteolytic digestion as an efficient tool for
the mass spectrometric identification of carbohydrate-
binding peptides in animal and plant lectins. In the final
elution step, competitive elution with a carbohydrate solu-
tion is generally suitable for specific isolation of ligand
epitopes; however, it requires high concentrations. Here,
we show that a wide range of organic-aqueous eluent mix-
tures are feasible as substitutes for elution [2, 3].
GalNAc and β-lactose were obtained from Sigma Aldrich
(Steinheim, Germany). Blood group A- and B-trisaccharides
and -tetrasaccharide were obtained from Carbosynth Ltd.
(Compton, UK). The blood group A- and B-trisaccharides were
conjugated and tested for bioactivity as described [3], then
conjugated to Sepharose beads to establish the affinity matrix
for galectin binding in the same manner, as carried out for
lactose [2].
Immobilized Carbohydrate Columns
Two hundred microliters drained Sepharose 4B was washed
with 1 mL 0.5 M Na2CO3 pH 11 and drained. The Sepharose
was resuspended in 300 mL of 0.5 M Na2CO3 and 40 μL
divinyl sulfone (DVS) was added dropwise. The suspension
was mixed end-over-end in a 90° rotary sample mixer for
70 min at 25 °C in the dark. The matrix was drained and
washed with 2 mL 0.5 M Na2CO3 pH 11 and resuspended in
200 μL of 1% carbohydrate solution in 0.5 M Na2CO3 pH 10
and mixed for 18 h at 25 °C. The affinity matrix was then
washed with 2 mL 0.5 M Na2CO3 pH 10. Blocking of
unreacted vinyl groups was carried out by incubation with
1 M ethanolamine pH 8.5 for 1 h at 25 °C. Next, the matrix
was washed sequentially with 0.1 M ammonium acetate con-
taining 0.5 M NaCl, pH 4, and 0.1 M Tris-HCl/0.5 M NaCl,
pH 8. Finally, the matrix was equilibrated in PBS buffer
(25 mM Na2HPO4/20 mM NaCl, pH 7.5) for hGal-3 binding
or HEPES buffer (10 mM HEPES, 25 mM NaCl2, 5 mM
CaCl2, 1 mM MnCl2, pH 7.4) for SBA binding and stored at
4 °C.
For control experiments, a Sepharose blank was prepared by
activating the matrix with DVS as described above. The matrix
was then treated with 1 M ethanolamine for 18 h at 25 °C and
washed with buffers using the same sequence as described
above.
In this study, we have applied the CREDEX-MS meth-
od using pressure-enhanced proteolysis to the identifica-
tion of ligand epitopes of blood group oligosaccharides to
human galectin-3 (hGal-3) and glycine max lectin (Soy-
bean Agglutinin, SBA). The results show specific epitope
peptides residing in the structures of lectins, which pro-
vide a molecular differentiation of oligosaccharide epi-
topes that should be applicable to a blood group determi-
nation assay.
Proteolytic Digestion
Due to the high resistance of lectins to enzymatic hydrolysis,
SBA was first denatured at 100 °C for 25 min prior to digestion.

Y. Baschung et al.: Oligosaccharide Epitopes in Lectins by Affinity-MS
Figure 1. (a) Proteolytic extraction mass spectrometric approach for identifying carbohydrate-binding structures in proteins. The
lectin is first digested in solution and the resulting fragments are bound on a column with immobilized carbohydrate. The non-binding
peptides are removed by washing and the binding fragments are subsequently eluted. (b) Chemical structures of the carbohydrate
ligands investigated: (C1) N-Acétylgalactosamine (2-acétamido-2-désoxy-D-galactopyranose), (C2) β-lactose (β-D-
galactopyranosyl-(1-4)-β-D-glucose), (C3) blood group A-trisaccharide (α-L-Fuc-(1-2)-[α-D-GalNAc-(1-3)]-β-D-Gal), (C4) blood group
B-trisaccharide (α-L-Fuc-(1-2)-[α-D-Gal-(1-3)]-β-D-Gal), (C5) blood group A-tetrasaccharide type 2 (α-D-GalNAc-(1-3)-[α-L-Fuc-(1-
2)]-β-D-Gal-(1-4)-β-D-GlcNAc)
A mixture of trypsin and chymotrypsin (enzyme/substrate ra-
tios 1:20 and 1:40, respectively) in Tris-buffer (100 mM Tris-
HCl, 10 mM CaCl2, pH 8.0) was used for SBA, while hGal-3
was digested using trypsin (enzyme/substrate ratio, 1:20) in
phosphate-buffered saline (PBS, 25 mM, pH 7.5) with 1 g/L
dithiothreitol (DTT). Proteolytic digestion was then performed
at high pressure (50 s at 40 kpsi, 10 s at atmospheric pressure)
for 120 cycles at 37 °C using a Barocycler 2320EXT (Pressure
Bioscience, Boston, MA, USA). A 1-μL aliquot sample from
the digestion mixture was then subjected to MALDI-MS
analysis.
Proteolytic Extraction-Mass Spectrometry
The SBA and hGal-3 digest mixtures were diluted 1:2, respec-
tively, using HEPES buffer and PBS prior to the addition onto
the affinity column containing immobilized carbohydrates.
After incubation for 12 h at 37 °C the supernatant was removed

Y. Baschung et al.: Oligosaccharide Epitopes in Lectins by Affinity-MS
and analyzed by MALDI-MS. Any traces of free peptides were
washing fractions did not provide any signals by MALDI-
MS. Elution with lactose-containing buffer was then performed
twice with 400 μL 0.3 M lactose in PBS (pH 7.5) while shaking
at 37 °C for 15 min. A surrogate elution system containing
acetonitrile was employed on an alternative set of samples, by
adding 400 μL ACN/0.1% TFA 2:1 onto the Sepharose slurry
and shaking at 37 °C for 15 min. This procedure was carried
out twice, and each time the eluate was collected.
washed out from the column with four times 400 μL HEPES
for SBA and PBS for hGal-3, and the washing fraction was
analyzed by MALDI-MS. The peptides bound to the
immobilized carbohydrate were eluted under shaking with
400 μL ACN/0.1% TFA (2:1) at 37 °C for 15 min, and the
procedure repeated twice. The elution fractions were then
combined, concentrated, and analyzed by MALDI-MS.
Mass Spectrometry
Proteolytic Excision-Mass Spectrometry
ESI mass spectra were obtained with a Bruker (Bruker
Daltonik, Bremen, Germany) Esquire 3000+ and a Bruker
HCT ion trap mass spectrometer. A Bruker Autoflex
Smartbeam mass spectrometer equipped with external fully
automated X-Y target stage MALDI source with pulsed colli-
sion gas was employed for MALDI-MS analyses. The pulsed
nitrogen laser was operated at 337 nm and ions generated by 10
laser shots were accumulated for 0.5–1 s at 30 V and extracted
at 15 V. Mass spectra were obtained using 10 laser shots for
each scan and accumulating 30–60 scans. The matrix solution
was prepared by dissolving 10 mg of 2,5-dihydroxybenzoic
acid in 1 mL acetonitrile/0.1% TFA in water (2:1 v/v). A 0.5-
μL aliquot of matrix solution was deposited on the MALDI
target, mixed with 0.5 μL of sample solution and allowed to dry
in ambient air. External calibration was performed using the
monoisotopic masses of singly protonated ions of human an-
giotensins I and II, bradykinin, neurotensin, bovine insulin ß-
chain (oxidized), and bovine insulin.
A solution of 20 μg SBA or hGal-3 in respectively 200 μL
HEPES or PBS buffer, pH 7.5, was added to 200 μL affinity
matrix and allowed to bind for 12 h at 37 °C. Any free
(unbound) lectin was washed out with 2 × 400 μL binding
buffer (with 1 g/L DTT) and the washing fractions were ana-
lyzed by MALDI-MS. The binding buffer was then switched to
Tris-buffer (with 5 mM MnCl2 added to allow carbohydrates to
interact with SBA) and proteolytic digestion of bound lectin
was performed by application to high-pressure cycles (30 kpsi
for 50 s, atmospheric pressure for 10 s, 50 °C, 90 cycles) with a
mixture of trypsin and chymotrypsin (1:20 and 1:30). Follow-
ing digestion, the supernatant was analyzed by MALDI-MS.
Free (unbound) peptides were washed out with 4 × 400 μL
binding buffer and the washing fractions analyzed by
MALDI-MS. The affinity-bound peptides were then eluted
with two times 200 μL ACN/0.1% TFA 2:1 at 37 °C for
15 min under shaking.
Aliquots of 1 μL of the sample solution and the saturated
matrix solution were mixed on the stainless steel MALDI target
and allowed to dry. Acquisition of spectra was carried out at an
acceleration voltage of 20 kV and a detector voltage of 1.5 kV.
Peptide Synthesis
Based on the identification of lactose and carbohydrate-binding
site peptides [2], three human galectin-3 peptides were synthe-
sized by SPPS: hGal-3(152–162), GNDVAFHFNPR (1);
hGal-3(177–183), LDNNWGR (2); hGal-3(130–144),
MLITILGTVKPNANR (3). The lactose carrier peptide, N-
Me-Aoa-Gly-Phe-Lys-Lys-Gly-COOH was synthesized and
lactosylated as previously described [2, 3]. All peptide synthe-
ses were carried out on a NovaSyn TGR resin according to the
Fmoc/tBu strategy [9, 10] using a semi-automated peptide
synthesizer (Intavis EPS 221; Köln, Germany). Preparative
RP-HPLC purification of the synthetic peptides were carried
out on a Knauer HPLC system (Bad Homburg, Germany)
using a GROM-SIL 120 ODS-4 SE HPLC column (Grom,
Herrenberg-Kayh, Germany). HPLC analyses were monitored
by UV detection at 220 nm. Sequence and homogeneities of all
peptides were confirmed by ESI-MS.
Determination of Affinity Binding Constants
Binding studies of synthetic peptides and full-length galectins
and glycine max lectin with lactose and GalNAc were per-
formed with a surface acoustic wave (SAW) biosensor (K5-
Ssens; SAW-Instruments, Bonn, Germany [11]; Nanotemper
GmbH, München, Germany).
A monolayer (SAM) of 16-mercaptohexadecanoic acid was
formed on the surface of the chip, as previously described [12].
The GalNAc-lactosyl-glycoprobe was immobilized on the
SAM by carboxyl-group activation with a 1:1 mixture (v/v) of
200 mM (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
(EDC) and 50 mM N-hydroxysuccinimide (NHS) [12]. A 10-
μM aqueous glycoprobe solution (pH 6.5) was used for immo-
bilization, followed by capping of unreacted carboxyl groups
with 1 M ethanolamine, pH 8.5. After changing the running
buffer to HEPES (pH 7.4) for SBA or PBS (pH 7.5) for hGal-3
and synthetic peptides, 150 μL solutions of increasing concen-
trations (0.1–1 mM PBS) of synthetic human lectin peptides
were injected on the chip with the immobilized glycoprobe at a
flow rate of 30 μL min−1. Intact lectins were injected at con-
centrations of 0.5–10 μM in buffer in their corresponding
buffer at the same flow rate. After each injection, the bound
Affinity Mass Spectrometry of Galectin-Derived
Peptides
For each of the synthetic peptides (s. Table 1), a solution of
30 μg peptide or lectin in PBS pH 7.5 was added over the
affinity column with carbohydrates (lactose, A-Tri, or B-Tri)
immobilized on DVS-activated Sepharose. After 12 h shaking
at 37 °C, the supernatant was analyzed by MALDI-MS. The
columns were then washed with binding solvent until the

Y. Baschung et al.: Oligosaccharide Epitopes in Lectins by Affinity-MS
Table 1. Summary of KD determinations between hGal-3 and synthetic hGal-3 peptides against lactose, and full-length SBA against GalNAc
No.
Peptide/protein
Immobilized carbohydrate
KD (mM)
R2
1
2
3
4
5
hGal-3(152–162)
hGal-3(177–183)
hGal-3(130–144)
hGal-3 full length
SBA full length
Lactose
Lactose
Lactose
Lactose
GalNac
11.7
2.4
0.97
0.99
n.a.
0.98
0.99
n.a.
4.8 × 10−3
25.5 × 10−3
peptide was left to dissociate for 5 min and the chip surface was
regenerated with 60% ACN, pH 6.5. After 5 min equilibration
time the next injection was allowed. After fitting the recorded
data according to the theoretical 1:1 Langmuir binding model,
the extracted observed rate constants (kobs = koff + Canalyte × kon)
were plotted versus the analyte concentrations. Linear regres-
sion of the data points yielded the association (kon) and disso-
ciation (koff) rate constants that were used to obtain the equi-
librium dissociation constant KD = koff × kon−1. The obtained
KD values are summarized in Table 1. A negative control for
determination of unspecific binding was performed by
injecting a solution of 0.5–20 μM myoglobin in PBS at iden-
tical conditions as described above.
[16]. Digestion times and enzymes/substrate ratios were opti-
mized as described in the BExperimental^ section and digestion
yields were estimated for all lectins by 1D gel electrophoresis.
Epitope Ligand Identification and Differentiation
of Blood Group Oligosaccharides in Lectins
Carbohydrates with different structural features (lactose;
GalNAc; blood group A- and B-trisaccharides; blood group
A-tetrasaccharide) were investigated as ligands (s. Fig. 1b).
SBA shows high affinities to GalNAc and galactose moieties
[17], which prompted us to study interactions with GalNac,
blood group A- and B-trisaccharides. MALDI-MS analyses of
the elution fractions upon proteolytic excision and extraction
from the affinity columns performed with immobilized
GalNAc showed ions corresponding to 3 specific peptide se-
quences (87–107) (1), (129–147) (2), and (206–224) (3)
(Fig. 3a). The identical epitope ligands were found in the
elution fractions from A- and B-trisaccharides, with an addi-
tional specific peptide sequence (108–125) (4) (Fig. 3b, c). The
epitope peptides (1), (2), and (3) identified in the elution
fractions contain the amino acid residues Asp-88, Gly-106,
Asn-130, and Asp-215 in direct contact with the carbohydrate
ligand, in complete agreement with the X-ray crystal structure
data (s. Fig. 6a). In the crystal structure, Rao et al. reported that
Asp-88 forms a bidentate hydrogen bond with C3-0H and C4-
0H hydroxyl groups, while the nitrogen group ND2 of Asn-130
and of Gly-106 form hydrogen bonds with C2-0H and C3-0H
hydroxyl groups, respectively, and the 4-0H hydroxyl group
forms another hydrogen bond with the backbone-NH of Leu-
214 [18]. The fourth peptide domain (4) identified in the
elution fractions of A- and B-trisaccharide include the loop
(112–119), where electron density has been found to be weak,
and is located close to the sugar binding site [18] (s. Fig. 6a).
The presence of (4) in the elution fraction can therefore be
explained by an interaction between the branched oligosaccha-
rides and the peptide loop.
Results and Discussion
Primary Structure Characterization of Lectins
by Proteolytic Peptide Mapping
As a first step in the identification of carbohydrate-binding
sites, the primary structures of the investigated lectins were
characterized by proteolytic peptide mapping and mass spec-
trometry, particularly to establish the conditions for the proteo-
lytic excision and extraction, and formation of regular, repro-
ducible peptide fragment mixtures. Native SBA has been pre-
viously reported to be highly resistant to enzymatic hydrolysis,
requiring heat treatment and multiple proteases for complete
digestion [13]. Cycles of high pressure during proteolytic di-
gestion uses alternating high hydrostatic pressure to facilitate
thermodynamic perturbation of molecular interactions and has
been shown to increase hydrolysis speed and recovery and
improve sequence coverage [14, 15]. The use of pressure-
enhanced proteolytic digestion provided a complete lectin di-
gestion within 1–2 h, whereas 12 h were necessary to obtain
comparable digestion results using a standard in-solution di-
gestion protocol at 37 °C and atmospheric pressure. A compar-
ison of the MALDI-MS data of SBA for both digestion
methods after 2 h showed the incomplete digestion of the in-
solution procedure with a calculated sequence coverage of 32%
compared to the 90% sequence coverage obtained with the
high-pressure digestion at 40 kpsi (Fig. 2). Nearly full sequence
coverage can be obtained with proteolytic peptide mapping on
both lectins, except for small sequences of 1–4 amino acids
(m/z < 600) and the Asp-75 glycosylated peptide (71–75) of
glycine max lectin (Fig. 2; Table S1, Supporting Information).
SBA is known to form C-terminal truncated isolectin structures
with 246 amino acids, which explains the cleavage at Ser246
An identical set of experiments provided the identification
of the carbohydrate recognition domains of hGal-3. MALDI-
MS analyses of the elution fractions upon proteolytic excision
and extraction from the affinity columns performed with
immobilized lactose and B-trisaccharide, containing affinity-
bound hGal-3 peptides, showed only two molecular ions cor-
responding to peptides (152–162) (1) and (177–183) (2). From
the corresponding experiments with immobilized A-tri- and A-
tetrasaccharides, the identical peptides (1) and (2) and a specific
additional third peptide (130–144) (3) were identified (Figs. 4a,

Y. Baschung et al.: Oligosaccharide Epitopes in Lectins by Affinity-MS
Figure 2. Comparison of MALDI-TOF mass spectra of SBA proteolytic digestion after 120 cycles of 50 s at 40 kpsi and 10 s at
atmospheric pressure and 37 °C (a) and standard proteolytic digestion at atmospheric pressure and 37 °C (b). Digestion procedures
were performed with a mixture of trypsin and chymotrypsin (enzyme/substrate ratios 1:20 and 1:40, respectively) in Tris-buffer
(100 mM Tris-HCl, 10 mM CaCl2, pH 8.0). All peptide fragments produced singly charged ions and are labeled T for tryptic and C for
chymotryptic cleavage products. In addition, peaks corresponding to sodium and potassium adducts ([M + Na+]+, [M + K+]+) were
observed in all spectra. The region with m/z > 1300 Da has been magnified
b and 6a). These results revealed that the human galectin-3
binding region for lactose and B-Tri is formed by amino acids
from the domains (152–162) and (177–183) (s. Fig. 4a). The
peptides (1) and (2) comprise the key amino acids that are in
direct contact with the ligand, in agreement with previous
results by X-Ray crystallography [19–21]. The majority of
the interactions are formed between the side-chains of
His158, Asn60, and Arg162 from peptide (1) and the galactose
subunit. In peptide (2), the residue W-183 participates through
its planar side-chain in a CH-π interaction with the B face of the
galactose ring. The side-chain of the W-183 residue is not only
involved in binding of the galactose ring but also determines its
orientation.
In addition to the peptides (152–162) and (177–183), the
recognition sites for A-tri and A-tetra include the peptide (130–
144) (Figs. 4b and 6b). The presence of this peptide in the
elution fraction is well explained by the Arg-144 residue which
is positioned to interact with other saccharide residues linked to
the galactose O-3 group [20–23]. In the case of A-tri- and A-
tetra-saccharide, the O-3-linked residue is α-D-GalNAc. It is
noteworthy that B-tri also has a subunit linked to the galactose
O-3, α-D-Gal; however, the third peptide, (130–144) was not
identified as a binding site for B-tri. These results suggest that
peptide 3 (130–144) is specific for α-D-GalNAc and does not
bind to α-D-Gal. To support this conclusion, affinity-MS ex-
periments were performed with a synthetic version of peptide
3, prepared by solid-phase peptide synthesis. In agreement with
the CREDEX-MS results, the peptide (130–144) indeed was
ascertained to bind only to A-tri- and A-tetrasaccharide, but did
not bind to B-trisaccharide or to lactose.
Elution Solvents for Dissociation of Carbohydrate--
Peptide Complexes
Elution of the affinity-bound peptides can be performed with a
concentrated solution of the identical carbohydrate
immobilized on the affinity column; the carbohydrate in solu-
tion thereby displacing the affinity-bound peptides from the
immobilized carbohydrate in a competitive manner. A different
carbohydrate may be used for elution if the affinity of the
protein or peptide is comparable to the immobilized carbohy-
drate. To prevent formation of cysteine- containing peptide
dimers through disulfide bonds, DTT (1 g/L) can be added to
the eluent without adverse effects. For the elution of affinity-

Y. Baschung et al.: Oligosaccharide Epitopes in Lectins by Affinity-MS
Figure 3. MALDI-TOF mass spectra of elution fractions from proteolytic excision of glycine max lectin form GalNAc (a), A-tri (b), and
B-tri (c) affinity columns. Peptides (1) correspond to SBA[87–107]; peptides (2) correspond to SBA[129–144], SBA[130–144], and
SBA[130–147]; peptides (3) correspond to SBA[206–224] and SBA[206–226]; and peptides (4) correspond to SBA[108–125] and
SBA[108–129]. In addition, peaks corresponding to sodium and potassium adducts ([M + Na+]+, [M + K+]+) were observed in all
spectra
bound galectin-3 peptides from 200 μL matrix with
immobilized lactose, the column was shaken with 400 μL
0.3 M lactose in 25 mM phosphate buffer with 25 mM NaCl,
pH 7.5 at 37 °C for 15 min, and this procedure repeated twice.

Y. Baschung et al.: Oligosaccharide Epitopes in Lectins by Affinity-MS
(a)
(b)
Figure 4. MALDI-TOF mass spectra of elution fractions from proteolytic excision of human galectin-3 form B-tri and A-tri columns.
(a) Peptides hGal-3(177–183) and hGal-3(152–162) were identified from the lactose and B-tri columns. (b) Peptides hGal-3(177–
183), hGal-3(152–162), and hGal-3(130–144) were identified from the A-tri and B-tri columns. Additional peaks corresponding to
sodium and potassium adducts ([M + Na+]+, [M + K+]+) were observed in all spectra
Due to the high concentration of carbohydrate required for
quantitative elution of affinity-bound peptides, sample cleaning
prior to MS analysis would be necessary even for HPLC-MS.
Since the cleaning step may lead to incomplete peptide recov-
ery, elution with aqueous-organic solvents would be advanta-
geous. Several combinations of TFA, HCl, acetic acid, THF,
ACN, and methanol were initially tested with varying results.
The most successful solvent systems were based on aceto-
nitrile, particularly ACN/0.1% TFA, 2:1 (v/v), pH 3, or 60–
80% ACN in water, pH 6. Elution was performed in the same
manner as for competitive elution, by shaking with 400 μL
eluent for 15 min and repeating twice this procedure. Since
acetonitrile-containing solutions had a dehydrating effect on
agarose-based affinity matrices depending on the percentage of
ACN, the matrix was equilibrated in the same buffer used for
protein binding immediately after the elution step. A 100%
ACN solution did not recover the bound peptides and
completely dehydrated and irreversibly damaged the affinity
matrix. Other elution systems such as 500 mM acetic acid or 5–
10% ACN in water did not perform well, and also solutions
based on non-volatile substances such as glycine or chaotropic
agents were found unsuitable. Elution from columns with
immobilized glycans containing sialic acid was effective at
acidic pH, but this rendered the column inactive for subsequent
applications due to cleavage of the sialic acid; however, this
effect could be completely prevented by using 70% ACN in
25 mM PBS, pH 7.5.
The above ACN-containing elution solvents were initially
developed with synthetic lectin peptides but proved equally
effective for full-length lectins. The efficacy of the organic
eluent solvents on full-length lectins was established using
1D-SDS-PAGE which allowed easy evaluation of the intensity
of gel bands of supernatant, washing, and elution fractions.
Affinity Mass Spectrometric and Biosensor Char-
acterization of Lectin-Derived Peptides
To evaluate the binding properties of the synthetic peptide
epitopes, peptides were added over Sepharose affinity columns
with the immobilized carbohydrate; following incubation, the
columns were washed until complete absence of ions detect-
able by mass spectrometry. Elution of affinity-bound peptides
was performed with the acetonitrile-based solvents described
above, and elution fractions analyzed by MALDI-TOF-MS.
Due to the complete lack of peptide ions in the washing
fractions, ion signals in the elution fraction corresponding to

Y. Baschung et al.: Oligosaccharide Epitopes in Lectins by Affinity-MS
(a)
(b)
Figure 5. KD determination for the complex between synthetic human galectin-3 peptides and lactose. (a) Fitted association and
dissociation curves of hGal-3(152–162) in a series of concentrations (0.2–0.8 mM) with the immobilized lactosyl-glycoprobe. (b) The
plot of kobs values versus peptide concentrations provided a KD of 11.7 mM for hGal-3(152–162) and 2.4 mM for hGal-3(177–183)
the peptide ascertain the affinity of the peptides to the carbo-
hydrate. In agreement with the results obtained by CREDEX-
MS, the peptides hGal-3(152–162) (1), hGal-3(177–183) (2),
and hGal-3(130–144) (3) all showed affinity towards lactose,
A-trisaccharide, and B-trisaccharide. Three different sets of
controls were performed for all peptides using Sepharose-
coupled maltose, Sepharose-coupled sucrose, and unmodified
Sepharose. None of the peptides exhibited affinity towards
maltose, sucrose, or to the unmodified Sepharose (data not
shown).
Dissociation constants KD were determined with the SAW
biosensor for the full-length galectin and SBA in complexes
Figure 6. (a) X-ray crystal structure of SBA in complex with a biantennary blood group antigen analog (PDB entry 1G9F) showing
SBA[87–107], SBA[129–147], and SBA[206–226] in red and MS-identified peptides specific for A-tri and B-tri, SBA[108–128], in blue.
Amino acids known to be in direct contact with the carbohydrate are highlighted in yellow. (b) X-ray crystal structure of human
galectin-3 CRD in complex with lactose (PDB entry 3ZSJ) showing hGal-3(152–162) and hGal-3(152–162) in red and MS-identified
peptides specific for A-tri and A-tetra, hGal-3(130–144), in blue. Amino acids known from the complex with lactose to be in direct
contact with the carbohydrate are highlighted in yellow

Y. Baschung et al.: Oligosaccharide Epitopes in Lectins by Affinity-MS
with lactose and GalNAc, respectively. The KD values were in
performance mass spectrometry with applications in life sci-
ence^ (MSLife). Partial support is also acknowledged from the
Bundesministerium für Wirtschaft (BMWi; SPR-MS).
the low micromolar range, in agreement with the values report-
ed for galectins using SPR [23, 24] and ITC [25]. The synthetic
peptide epitopes had significantly lower affinities for lactose
(approximately 1 to 14 mM) than the full-length proteins,
which is well explained as the small peptides represent just a
section of the complete CRD structure, respectively. Repetitive
measurements showed a reproducibility of 2 to 5% depending
the on the level of affinities measured. The two human peptides
hGal-3(177–183) and hGal-3(152–162) showed KD values of
2.4 and 11.7 mM, respectively (Figs. 5 and 6). Shortening of
the sequence (152–162) to (157–163) led only to a slight
decrease in affinity (KD 13.8 mM), while sequence prolonga-
tion to (157–175) to include Val171 and Asn174 residues
provided a 20-fold increase in affinity (KD 0.7 mM).
References
1. Lobsanov, J.D., Gitt, M.A., Leffler, H., Barondes, S.H., Rini, J.M.: X-ray
crystal structure of the human dimeric S-lac lectin, L-14-II, in complex
with lactose at 2.9 A resolution. J. Biol. Chem. 268, 27034–27038 (1993)
2. Moise, A., Andre, S., Eggers, F., Krzeminski, M., Przybylski, M., Gabius,
H.J.: Toward bioinspired galectin mimetics: identification of ligand-
contacting peptides by proteolytic-excision mass spectrometry. J. Am.
Chem. Soc. 133, 14844–14847 (2011)
3. Jimenez-Castells, C., Defaus, S., Moise, A., Przbylski, M., Andreu, D.,
Gutierrez-Gallego, R.: Surface-based and mass spectrometric approaches
to deciphering sugar-protein interactions in a galactose-specific aggluti-
nin. Anal. Chem. 84, 6515–6520 (2012)
4. Stefanescu, R., Born, R., Moise, A., Ernst, B., Przybylski, M.: Epitope
structure of the carbohydrate recognition domain of Asialoglycoprotein
receptor to a monoclonal antibody revealed by high-resolution proteolytic
excision mass spectrometry. J. Am. Soc. Mass Spectrom. 22, 148–157
(2011)
5. Stefanescu, R., Iacob, R.E., Damoc, E.N., Marquardt, A., Amstalden, E.,
Manea, M., Perdivara, I., Maftei, M., Paraschiv, G., Przybylski, M.: Mass
spectrometric approaches for elucidation of antigenantibody recognition
structures in molecular immunology. Eur. J. Mass. Spectrom. 13, 69–75
(2007)
6. Iurascu, M.I., Marroquin-Belaunzanar, O., Petrausch, U., Renner, C.,
Przybylski, M.: An HLA-B27 homodimer specific antibody recognozes
a discontinuous mixed-disulfide epitope identified by affinity-mass spec-
trometry. J. Am. Soc. Mass Spectrom. 27, 1105–1112 (2016)
7. Juszczyk, P., Paraschiv, G., Szymanska, A., Kolodziejczyk, A.S.,
Rodziewicz-Motowidlo, S., Grzonka, Z., Przybylski, M.: Binding epi-
topes and interaction structure of the neuroprotective protease inhibitor
cystatin C with beta-amyloid revealed by proteolytic excision mass
spectrometry and molecular docking simulation. J. Med. Chem. 52,
2420–2428 (2009)
8. Krzeminski, M., Singh, T., Andre, S., Lensch, M., Wu, A.M., Bonvin,
A.M., Gabius, H.J.: Human galectin-3 (Mac-2 antigen): defining molec-
ular switches of affinity to natural glycoproteins, structural and dynamic
aspects of glycan binding by flexible ligand docking and putative regu-
latory sequences in the proximal promoter region. Biochim. Biophys.
Acta. 1810, 150–161 (2011)
9. Merrifield, R.B.: Solid-phase peptide synthesis. 3. An improved synthesis
of bradykinin. Biochemistry. 3, 1385–1390 (1964)
10. Carpino, L.A., Han, G.Y.: 9-Fluorenylmethoxycarbonyl function, a new
base-sensitive amino-protecting group. J. Am. Chem. Soc. 92, 5748–
5749 (1970)
11. Gronewold, T.M.: Surface acoustic wave sensors in the bioanalytical field:
recent trends and challenges. Anal. Chim. Acta. 603, 119–128 (2007)
12. Drǎguşanu, M., Petre, B.A., Przybylski, M.: Epitope motif of an anti-
nitrotyrosine antibody specific for tyrosine-nitrated peptides revealed by a
combination of affinity approaches and mass spectrometry. J. Pept. Sci.
17, 184–191 (2011)
13. Ma, Y., Wang, T.: Deactivation of soybean agglutinin by enzymatic and
other physical treatments. J. Agric. Food Chem. 58, 11413–11419 (2010)
14. López-Ferrer, D., Petritis, K., Hixson, K.K., Heibeck, T.H., Moore, R.J.,
Belov, M.I., Camp, D.G., Smith, R.D.: Application of pressurized sol-
vents for ultrafast trypsin hydrolysis in proteomics: proteomics on the fly.
J. Proteome Res. 7(8), 3276–3281 (2008)
Conclusions
The combination of pressure-assisted enzymatic hydrolysis,
specific proteolytic extraction/excision of carbohydrate-
binding peptides, and mass spectrometric analysis of the eluted
peptides is shown here to be a highly efficient tool for identi-
fying and differentiating specific CRD structures. Pressure-
assisted enzymatic hydrolysis showed an increased digestion
efficiency of enzyme-resistant lectins. Despite the relatively
weak interactions between peptides and carbohydrates as illus-
trated by the relatively high KD values, we demonstrate here the
specific differentiation between the recognition structures to
carbohydrates at the submolecular level, ascertained by the
binding of natural and synthetic peptides of hGal-3. The only
difference between A-trisaccharide and B-trisaccharide, the N-
acetylation of A-tri, is also the marker that differentiates blood
group A from blood group B types. The remarkable capability
of the peptide hGal-3(130–144) to differentiate between these
two carbohydrate structures may render peptide epitopes a
potential alternative to much larger blood group A determinant
proteins, such as the Dolichos biflorus (anti-A1) [20]. While
the single peptide hGal-3(130–144) cannot have agglutinating
properties, branched polypeptide bioconjugates [26, 27]
exhibiting multiple peptide ligand copies may lead to well
amenable alternatives to lectins and human sera as blood-
typing reagents. These results indicate the potential of
pressure-assisted proteolytic excision- and extraction-mass
spectrometry for identifying affinity-derived lectin epitope pep-
tides, with the potential to be valuable therapeutic agents, or
reagents for investigating cell surface carbohydrates and asso-
ciated pathophysiological processes.
Acknowledgements
We thank Drs. Stefan Maeser and Elisa Peroni for the valuable
discussions and critical reading of the manuscript.
15. Balny C. Biochimica et Biophysica Acta-Proteins and Proteomics 1764
(2006) 632–639
16. Sharon, N., Lis, H.: Detection, occurrence and isolation. In: Sharon, N.,
Lis, H. (eds.) Lectins, 2nd edn, pp. 33–62. Kluwer Academic Publishers,
Dordrecht, Boston, MA (2003a)
17. Sharon, N., Lis, H.: Specificity and affinity in lectins. In: Sharon, N., Lis,
H. (eds.) , 2nd edn, pp. 63–104. Kluwer Academic Publishers, Dordrecht,
The Netherlands; Boston, MA (2003)
Funding Information
This work has been partially supported by the European Union
through the Marie-Curies IRSES grant BIntegrating high

Y. Baschung et al.: Oligosaccharide Epitopes in Lectins by Affinity-MS
18. Rao, V.S.R., King, L., Pradman, K.: Three dimensional structure of the
soybean agglutinin-Gal/GalNAc complexes by homology modeling. J.
Biomol. Struct. Dyn. 15(5), 853–860 (1998)
19. Seetharaman, J., Kanigsberg, A., Slaaby, R., Leffler, H., Barondes, S.H.,
Rini, J.M.: X-ray crystal structure of the human galectin-3 carbohydrate
recognition domain at 2.1-A resolution. J. Biol. Chem. 273, 13047–13052
(1998)
20. Saraboji, K., Håkansson, M., Genheden, S., Diehl, C., Qvist, J.,
Weininger, U., Nilsson, U.J., Leffler, H., Ryde, U., Akke, M., Logan,
D.T.: The carbohydrate-binding site in galectin-3 is preorganized to
recognize a sugarlike framework of oxygens: ultra-high-resolution struc-
tures and water dynamics. Biochemistry. 51, 296–306 (2011)
21. Diehl, C., Engström, O., Delaine, T., Håkansson, M., Genheden, S.,
Modig, K., Leffler, H., Ryde, U., Nilsson, U.J., Akke, M.: Protein
flexibility and conformational entropy in ligand design targeting the
carbohydrate recognition domain of galectin-3. J. Am. Chem. Soc. 132,
14577–14589 (2010)
sequence analysis, homology modeling and mutagenesis studies of ham-
ster galectin-3. Glycobiology. 8, 45–57 (1998)
23. Murakami, T., Yoshioka, K., Sato, Y., Tanaka, M., Niwa, O., Yabuki, S.:
Synthesis and galectin-binding activities of mercaptododecyl glycosides
containing a terminal beta-galactosyl group. Bioorg. Med. Chem. Lett.
21, 1265–1269 (2011)
24. Stowell, S.R., Arthur, C.M., Mehta, P., Slanina, K.A., Blixt, O., Leffler,
H., Smith, D.F., Cummings, R.D.: Galectin-1, -2, and -3 exhibit differ-
ential recognition of sialylated glycans and blood group antigens. J. Biol.
Chem. 283, 10109–10123 (2008)
25. Bachhawat-Sikder, K., Thomas, C.J., Surolia, A.: Thermodynamic anal-
ysis of the binding of galactose and poly-N-acetyllactosamine derivatives
to human galectin-3. FEBS Lett. 500, 75–79 (2001)
26. Liu, J., Gray, W.D., Davis, M.E., Luo, Y.: Peptide- and saccharide-
conjugated dendrimers for targeted drug delivery: a concise review.
Interface Focus. 2, 307–324 (2012)
27. Xiao, S., Abu-Esba, L., Turkyilmaz, S., White, A.G., Smith, B.D.: Mul-
tivalent dendritic molecules as broad spectrum bacteria agglutination
agents. Theranostics. 3, 658–666 (2013)
22. Henrick, K., Bawumia, S., Barboni, E.A., Mehul, B., Hughes, R.C.:
Evidence for subsites in the galectins involved in sugar binding at the
nonreducing end of the central galactose of oligosaccharide ligands: