Mass spectrometric method for determining the uronic acid epimerization in heparan sulfate disaccharides generated using nitrous acid

Article abstract of DOI:10.1021/ac3016054

Heparan sulfate (HS) glycosaminoglycans (GAGs) regulate a host of biological functions. To better understand their biological roles, it is necessary to gain understanding about the structure of HS, which requires identification of the sulfation pattern as well as the uronic acid epimerization. In order to model HS structure, it is necessary to quantitatively profile depolymerization products. To date, liquid chromatography-mass spectrometry (LC-MS) methods for profiling heparin lyase decomposition products have been shown. These enzymes, however, destroy information about uronic acid epimerization. Deaminative cleavage using nitrous acid (HONO) is a classic method for GAG depolymerization that retains uronic acid epimerization. Several chromatographic methods have been used for analysis of deaminative cleavage products. The chromatographic methods have the disadvantage that there is no direct readout on the structures producing the observed peaks. This report demonstrates a porous graphitized carbon (PGC)-MS method for the quantification of HONO generated disaccharides to obtain information about the sulfation pattern and uronic acid epimerization. Here, we demonstrate the separation and identification of uronic acid epimers as well as geometric sulfation isomers. The results are comparable to those expected for benchmark HS and heparin samples. The data demonstrate the utility of PGC-MS for quantification of HS nitrous acid depolymerization products for structural analysis of HS and heparin.

Full text of DOI:10.1021/ac3016054

Article
pubs.acs.org/ac
Mass Spectrometric Method for Determining the Uronic Acid
Epimerization in Heparan Sulfate Disaccharides Generated Using
Nitrous Acid
Vanessa Leah Gill, Qi Wang, Xiaofeng Shi, and Joseph Zaia*
Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts, United States
S
* Supporting Information
ABSTRACT: Heparan sulfate (HS) glycosaminoglycans
(GAGs) regulate a host of biological functions. To better
understand their biological roles, it is necessary to gain
understanding about the structure of HS, which requires
identification of the sulfation pattern as well as the uronic acid
epimerization. In order to model HS structure, it is necessary
to quantitatively profile depolymerization products. To date,
liquid chromatography−mass spectrometry (LC-MS) methods
for profiling heparin lyase decomposition products have been
shown. These enzymes, however, destroy information about uronic acid epimerization. Deaminative cleavage using nitrous acid
(HONO) is a classic method for GAG depolymerization that retains uronic acid epimerization. Several chromatographic
methods have been used for analysis of deaminative cleavage products. The chromatographic methods have the disadvantage that
there is no direct readout on the structures producing the observed peaks. This report demonstrates a porous graphitized carbon
(PGC)-MS method for the quantification of HONO generated disaccharides to obtain information about the sulfation pattern
and uronic acid epimerization. Here, we demonstrate the separation and identification of uronic acid epimers as well as geometric
sulfation isomers. The results are comparable to those expected for benchmark HS and heparin samples. The data demonstrate
the utility of PGC-MS for quantification of HS nitrous acid depolymerization products for structural analysis of HS and heparin.
eparan sulfate (HS) and heparin glycosaminoglycans
is composed of five consecutive N- and 2,6-di-O-sulfated
H(GAGs) have been identified as key players in several disaccharide repeats.10
physiological and pathophysiological processes.1−4 These
GAGs are structurally related linear polysaccharides. They are
biosynthesized as repeating units of glucuronic acid and N-
acetyl-glucosamine, [4-GlcAβ-1,4-GlcNAcα1-]n.5 The nascent
chains are modified through a series of enzymatic reactions
initiated by N-deacetylation and N-sulfation of GlcNAc
residues, followed by epimerization of some D-glucuronic acid
to L-iduronic acid (IdoA) and subsequent sulfation at the C2
position. In the mature chains, GlcN units may be N-acetylated
(GlcNAc), N-sulfated (GlcNS), or (rarely) unsubstituted
(GlcN) and may be sulfated at the C6 and, rarely, C3
positions. Because of these modifications, HS and heparin
chains are composed of disaccharide units with unique patterns
of N-acetylation, N-sulfation, O-sulfation, and uronosyl
epimerization. Mature chains have domains of high content
of IdoA and sulfation, interspersed with unmodified domains
and those of intermediate sulfation. The expression of HS is
required for embryogenesis and for normal functioning of every
adult physiological system.6,7 Although the biological roles of
HS and heparin are highly diverse, it has been shown that their
functions depend largely upon the proteins to which they
interact as determined by the HS chain structures.8−10 For
example, binding is dependent on either 2-O- or 6-O-sulfation
patterns for FGF signaling in Drosophila tracheal development.8
The binding region for lipoprotein lipase in endothelial cell HS
Structural analysis of HS and heparin is necessary to
determine their biological activities, but experiments have
been limited by the extreme heterogeneity in their composition,
sulfation, acetylation, and epimerization patterns. Many
methods focus on enzymatic depolymerization of HS and
heparin followed by disaccharide analysis using liquid
chromatography (LC),11 capillary electrophoresis (CE),12
MS,13 CE-MS,14 or LC-MS.15,16 However, the enzymatic
digestion cleaves glycosidic GlcN-HexA bonds, resulting in
loss of asymmetry at C5 of the original uronic acid because of
the generation of unsaturated bonds between C4 and C5.
An alternative method of depolymerization is hydrazinolysis
followed by deaminative cleavage using nitrous acid (HONO)
treatment. The deacetylation and deaminative cleavage results
in disaccharide units composed of uronic acid (GlcA or IdoA)
and 2,5-anhydromannose (aMan) bearing an aldehyde
group.17,18 The advantage of this classic method for GAG
depolymerization is that the original epimeric nature of the
uronic acid is retained. The resulting disaccharides have been
analyzed by paper electrophoresis or chromatography,16 and
anion exchange HPLC19 after reduction with NaB3H4, ion pair
Received: June 20, 2012
Accepted: August 7, 2012
Published: August 7, 2012
© 2012 American Chemical Society
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reverse phase LC (IPRP-LC) after coupling with para-
nitrophenyl-hydrazine,20 and IPRP-LC-MS after reduction
with NaBH4.21
Healthcare (Piscataway, NJ). The samples were separated using
a Waters Acquity Ultra Performance LC system (Milford, MA).
Disaccharides eluting from the LC column were analyzed using
an Applied Biosystems QSTAR Pulsar-I (Q-TOF) mass
spectrometer (Carlsbad, CA) operating in negative ion mode.
Solvents, gradients, and MS conditions used are described in
detail in the Supporting Information.
Tandem Mass Spectrometry. All tandem mass spectra
were obtained using a 12 T Fourier transform ion cyclotron
resonance (FTICR) mass spectrometer (SolariX, Bruker
Daltonics, Billerica, MA) operated in negative ion mode. MS
parameters used for the electron detachment dissociation
(EDD) and electron induced dissociation (EID) are described
in the Supporting Information.
Recently, a bioinformatics algorithm, ChamP, has been
developed to model HS chain structure based on compositional
analysis data.22 This algorithm requires disaccharide composi-
tional profiles from both lyase digested and deaminative
cleavage methods. However, in order to correctly model HS
structure from compositional analysis data, effective means of
analysis of deaminative cleavage products have to be used. As
shown by Kuberan’s group,21 IPRP-LC-MS is useful for
estimating C5-epimerase activity by resolving GlcA from
IdoA containing HONO disaccharides. Complete LC-MS
analysis of all products from deaminative cleavage is desirable
because the MS dimension provides direct validation to all
chromatographic peaks. Another major advantage of LC-MS is
that use of tandem MS provides structural information on peak
identities. Porous graphitized carbon (PGC) chromatography
has the potential to be well suited for the separation of all
HONO-derived product disaccharides (dp2). The high
isomeric resolving power23−26 of graphitized carbon avoids
the use of derivatization, which can be time-consuming, costly,
and usually decreases the sensitivity of analysis. The use of salts
or amine ion pairs in the mobile phase is not necessary, making
PGC well suited for interface with mass spectrometry. This
approach has been applied successfully to glycan classes
including O-linked, N-linked, and GAGs.27,28 PGC-LC-MS
under negative ionization conditions has been applied to the
analysis of enzymatically derived-dp2s from hyaluronan, keratan
sulfate, chondroitin sulfate, heparin, and HS.29,30 To date, an
LC-MS method for quantification of all deaminative cleavage
products of HS has not been described. This work shows PGC-
LC-MS for analysis of the complete range of HONO generated
disaccharides to obtain information about the sulfation pattern
and uronic acid epimerization. An effort was made to validate
the identifications of all disaccharide peaks using advanced
tandem mass spectrometry.
RESULTS AND DISCUSSION
■
Separation of HONO-Derived Disaccharides. Samples
of HS and heparin were subjected to N-deacetylation by
hydrazinolysis and deaminative cleavage by nitrous acid. These
conditions led to cleavage at GlcN as well as GlcNS units,
resulting in depolymerization to HexA-2,5-anhydromannose
(HexA-aMan) disaccharide units for completely N-deacetylated
HS chains (structures are shown in Supporting Information
Figure S-1 and the nitrous acid reaction in Supporting
Information Figure S-2). Reduction of the products with
NaBH4 followed by SEC-MS revealed the presence of a low
abundance of tetrasaccharides (<10% of the total products) in
addition to disaccharides. These tetrasaccharides are due
primarily to a side reaction of deaminative ring contraction.33
Disaccharides consistently accounted for >90% of the total
products and are considered to reflect the overall composition
of the HS and heparin samples. The structures of these
HONO-derived dp2s are shown in the Supporting Information
Figure S-3. Extracted ion chromatograms (EIC) for the
nonsulfated (m/z 339), monosulfated (m/z 419), and
disulfated (m/z 499) HexA-aManR disaccharides derived from
HONO treatment of HS from bovine kidney (HSBK), porcine
intestinal mucosa (HSPIM), and heparin separated by PGC are
shown in Figure 1. A peak corresponding to loss of sulfate in
the MS ion source was observed for the disulfated disaccharide.
Note that HSBK as well as other HS samples (see Supporting
Information Figure S-4) do not exhibit peaks 4 and 9. This
suggests that these peaks are specific to heparinoids from
porcine intestinal mucosa. Mass spectra for each HONO-
derived disaccharide from HSPIM are shown in Supporting
Information Figure S-5. A peak was observed for each
disaccharide [M − H]− ion. Low abundance peaks correspond-
ing to loss of SO3 were also observed in the mass spectra of the
disulfated disaccharides (peaks 8 and 9). The relative quantities
of the nine chromatographically separated HexA-aManR
disaccharides extracted from the PGC-MS data are summarized
in Supporting Information Table S-1. The identities of these
peaks will be discussed in subsequent sections. HSPIM and
heparin yielded predominantly disulfated disaccharides. This is
expected as the N- and 2,6-di-O-sulfated structure, IdoA(2S)-
GlcNS(6S), is generally the most abundant disaccharide unit in
heparin preparations.34 In contrast, HSBK generated more
abundant nonsulfated HexA-aManR dp2s.
EXPERIMENTAL SECTION
■
Hydrazinolysis and Nitrous Acid (HONO) Depolyme-
rization. Intact polysaccharides were deacetylated and
depolymerized according to Guo and Conrad.18 Details are in
the Supporting Information.
Enzymatic Digestion. HS from bovine kidney (HSBK)
and porcine intestinal mucosa (HSPIM) as well as heparin were
digested as previously described.14 Details are in the Supporting
Information.
Chemical O−Desulfation. 6-O-Desulfated disaccharides
were prepared according to Takano et al.31 2-O-Desulfated
disaccharides were prepared according to Jaseja et al.32 Details
are in the Supporting Information.
Exoglycosidase Digestion. The HONO-derived dp2s (1
μg) were treated with the exoenzymes at 37 °C for 2−24 h in a
final volume of 20 μL of 50 mM sodium acetate buffer, pH 4.5,
containing 100 μg/mL bovine serum albumin (BSA). Final
enzyme concentration for effective complete digestion was 500
milliunits/mL. Enzyme digests were terminated by heating the
solutions at 100 °C for 5 min.
Liquid Chromatography/Mass Spectrometry. The
PGC column (Hypercarb Column, 5 μm, 150 mm × 4.6
mm) was purchased from Thermo Electron Corporation
(Waltham, MA). The size exclusion chromatography (SEC)
column (Superdex peptide PC 3.2/30) was purchased from GE
The HS and heparin samples were also digested with a
mixture of heparin lyases I, II, and III, resulting in complete
depolymerization of the GAG chains to disaccharides (the
structures for which are shown in Supporting Information
Figure S-3). The codes used for the dp2s derived from enzyme
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and PGC-MS method for disaccharide analysis of HS and
heparin samples.
Identification of Uronic Acids. In the HS samples, there
are two peaks for the nonsulfated disaccharide with m/z 339.1
(peaks 1 and 2 in Figure 1). In order to identify the GlcA-
aManR, K5 polysaccharide was subjected to hydrazinolysis and
deaminative cleavage followed by PGC-MS. K5 polysaccharide,
a polymer consisting of unsulfated GlcAβ(1→4)GlcNAc
disaccharide units, showed one major peak at ∼9.8 min (Figure
2A). On the other hand, the EIC of m/z 339.1 from epimerized
and N-sulfated K5 polysaccharide (50% epimerization of the
HexA C5 position) showed two equally abundant peaks, peak 1
at 9.8 min and peak 2 at 10.2 min (Figure 2A). These data
indicate that the first and second peaks correspond to GlcA-
aManR and IdoA-aManR, respectively.
The exoglycosidases, β-glucuronidase and α-iduronidase,
were used to determine which HONO-derived dp2s have
GlcA and IdoA, respectively. β-Glucuronidase and α-
iduronidase catalyzes the hydrolysis of β-D-glucuronic acid
and α-L-iduronic acid residues, respectively, from the non-
reducing end of GAGs such as HS.41 These enzymes have been
shown to have very high specificity for unsulfated hexuronic
acid residues at the nonreducing end.42,43 These exoglycosi-
dases will, thus, have no activity on highly sulfated disaccharides
and will have no effect on the disulfated HONO-derived dp2s
containing 2-O-sulfate.
To confirm that peak 2 corresponds to IdoA-aManR, the
HONO-derived disaccharides from epimerized and N-sulfated
K5 polysaccharide were treated with α-iduronidase. Figure 2B
shows that there was ∼70% decrease in the second peak of
epimerized and N-sulfated K5 polysaccharide after treatment
with the enzyme. This verifies that the second peak is IdoA-
aManR.
The EIC of the monosulfated dp2 (m/z 419.1) from HSBK
showed four distinct peaks, whereas HSPIM and heparin
showed five peaks (peaks 3−7, Figure 1). After treatment with
β-glucuronidase, the intensity of peak 6 for HSBK and HSPIM
decreased by about 50% and 40%, respectively (Figure 2C,D).
β-Glucuronidase is specific for GlcA-(β1,4)/(β1,3)-HexNY6S/
3S where Y is either an acetyl or sulfate group.38 Consistent
with this report, β-glucuronidase did not have an effect on the
nonsulfated GlcA-aManR. Peak 7 for HSBK and HSPIM
showed ∼70% and 55% decrease, respectively, after treatment
with α-iduronidase (Figure 2E,F). In addition, there is a small
decrease (∼15% and 8% for HSBK and HSPIM, respectively)
in IdoA-aManR which is consistent with the findings of
Freeman et al.43 α-Iduronidase has greater activity for IdoA-
aMan(6S)R compared to IdoA-aManR. Peak 1′ represents the
resulting aMan(6S)R residue with m/z 243. These results
indicate that peaks 6 and 7 are GlcA-aMan(6S)R and IdoA-
aMan(6S)R, respectively.
Identification of Positional Sulfation Isomers. Chem-
ical O-desulfation was used to determine the positions of the
sulfates in the mono- and disulfated HONO-derived dp2s.
Selective 2-O-desulfation was achieved by treatment of HS and
heparin with alkali.32 By lyophilization, the 2-O-sulfated
hexuronic acid, quantitatively converted into an epoxidated
structure after treatment with a base such as NaOH, is
converted to a 2-O-desulfated HexA.44 Figure 3 shows that the
monosulfated peak 5 disappeared and the disulfated peak 8
decreased significantly after 2-O-desulfation for HSBK, HSPIM,
and heparin. There was a corresponding increase in IdoA-
aManR, indicating that peak 5 is IdoA(2S)-aManR. There was
Figure 1. Extracted ion chromatograms of HONO-derived dp2s
analyzed by PGC-MS from (A) HSBK, (B) HSPIM, and (C) heparin.
Peaks 1 and 2 are nonsulfated, peaks 3−7 are monosulfated, and peaks
8 and 9 are disulfated dp2s. For 8 and 9, the EIC for in-source loss of
SO3 is overlaid.
digestion are based on the nomenclature proposed by Lawrence
et al.35 Separation of these 4,5-unsaturated disaccharides was
performed by SEC-MS (see Supporting Information Figure S-
6). This method has been used extensively and successfully by
our group for disaccharide analysis.15,36−40 A comparison of the
deaminative cleavage in conjunction with PGC-MS and
enzymatic digestion in combination with SEC-MS is shown
in Supporting Information Table S-2. The two methods are
compared for equivalent disaccharide units based on degree of
sulfation. The two methods generated similar percentages for
each group of disaccharides. The enzymatic digestion/SEC-MS
method resulted in slightly higher percentages of monosulfated
dp2s. Incomplete hydrazinolysis or deaminative cleavage and
the presence of low abundance tetrasaccharides from
deaminative ring contraction may account for these minor
differences. Overall, the results from the two methods are in
good agreement, thus confirming the validity of the HONO
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Figure 2. Effect of exoglycosidases on HONO-derived dp2s. (A) EIC of the nonsulfated dp2 from K5 polysaccharide (solid line). The EIC of the
epimerized and N-sulfated K5 polysaccharide is shown in dashed lines. (B) EIC of the nonsulfated dp2 of epimerized and N-sulfated K5
polysaccharide with (dashed line) and without (solid line) treatment with α-iduronidase. After treatment with the enzyme, there is a decrease in
intensity of peak 2, as indicated by the gray arrow. After treatment with β-glucuronidase, the monosulfated peak 6 of (C) HSBK and (D) HSPIM
decreased. The monosulfated peak 7 of (E) HSBK and (F) HSPIM decreased significantly after incubation with α-iduronidase.
also an increase in the monosulfated peak 7. This is likely a
result of the removal of 2-O-sulfate from the disulfated peak 8.
Chemical 6-O-desulfation was achieved using a silylating
agent, MTSTFA, which has a high regioselectivity for 6-O-
desulfation.31 A report showed that, while this method can
remove 6-O-sulfates completely, it can also remove 2-O-sulfates
to a lesser degree.45 PGC-MS revealed that monosulfated peaks
3, 4, 6, and 7 as well as disulfated peaks 8 and 9 decreased in
intensity after treatment with MTSTFA (Figure 4). Together
with the results after treatment with exoglycosidases (Figure
2C-2F), this suggests that peaks 6 and 7 are GlcA-aMan(6S)R
and IdoA-aMan(6S)R, respectively. The peaks for the non-
sulfated dp2s, peak 1 GlcA-aManR and peak 2 IdoA-aManR,
increased after 6-O-desulfation. This is due to the removal of 6-
O-sulfates from the monosulfated dp2s. There was also an
increase in peak 5, IdoA(2S)-aManR, which can be due to the
removal of 6-O-sulfate from either peaks 8 or 9.
Arixtra, a pentasaccharide consisting of GlcNS(6S)-GlcA-
GlcNS(3S)(6S)-IdoA(2S)-GlcNS(6S)OCH3, was used to
identify the disulfated peaks 8 and 9 present in HSPIM and
heparin. Deaminative cleavage followed by PGC-MS revealed
three major and three minor cleavage products (Figure 5). The
first major peak 1′ corresponds to aMan(6S)R with m/z 243.
The three minor peaks (∼10% of total products) are
monosulfated dp2s and have the same retention times as
peaks 3, 4, and 5 of the HS and heparin samples. The last two
peaks are disulfated peaks with the same retentions times as
peaks 8 and 9. Arixtra was treated with Sulf2, an endosulfatase
that selectively removes 6-O-sulfates from HS chains. Previous
studies have revealed that Sulf2 acts predominantly on
HexA(2S)-GlcNS(6S) disaccharides and weakly on HexA-
GlcNS(6S).46,47 Figure 5B shows that the first disulfated peak 8
decreased in intensity after treatment with Sulf2, indicating that
this peak is IdoA(2S)-aMan(6S)R. The HONO-treated Arixtra
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Figure 3. Effect of chemical 2-O-desulfation on HONO-derived dp2s
from (A) HSBK, (B) HSPIM, and (C) heparin. The EIC of the
disaccharides after 2-O-desulfation are shown in dotted lines. For all
the samples, there is a decrease in monosulfated peak 5 and disulfated
peak 8 after treatment with NaOH and lyophilization (gray arrows).
There is also an increase in the nonsulfated peak 2 and monosulfated
peak 7 (white arrows).
Figure 4. Effect of chemical 6-O-desulfation on HONO-derived dp2s
from (A) HSBK, (B) HSPIM, and (C) heparin. The EIC of the
disaccharides after incubation with MTSTFA are shown in dotted
lines. For all the samples, there is a decrease in monosulfated peaks 3,
4, 6, and 7 as well as in the disulfated peaks 8 and 9 after 6-O-
desulfation (gray arrows). There is also an increase in both nonsulfated
peaks 1 and 2 and monosulfated peak 5 (white arrows).
was also subjected to chemical O-desulfation. Only peak 8
decreased after 2-O-desulfation (Figure 5C), which confirms
that this peak is IdoA(2S)-aMan(6S)R. As expected, there was
an increase in the peak at ∼32 min, the same retention time as
IdoA-aMan(6S)R, peak 7, from the HS samples and heparin. In
contrast, both disulfated peaks 8 and 9 disappeared after 6-O-
desulfation (Figure 5D). There was an increase in the peak at
∼16.5 min, the same retention time as the IdoA(2S)-aManR,
peak 5, from the HS samples. This is due to the removal of 6-
O-sulfate from the first disulfated peak 8, IdoA(2S)-aMan(6S)R.
These results indicate that the second disulfated peak 9 is, thus,
GlcA-aMan(3S)(6S)R. The results also revealed that the
treatment with MTSTFA removes both 3-O- and 6-O-sulfates
at the same rate. The 6-O-desulfation was performed at
different temperatures and reaction times to determine the
optimum conditions for selective removal of either 3-O- and 6-
O-sulfates (data not shown). However, selective removal was
not obtained at any of the conditions used.
Analysis of Pure Monosulfated HONO-Derived Dis-
accharides. Since no standards for the monosulfated dp2s
generated using deaminative cleavage were available, each
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Figure 5. Identification of disulfated dp2s. (A) EIC of Arixtra HONO disaccharides. (B) The EIC of the disaccharides after Sulf2 treatment are
shown in dotted lines. There is a decrease in the disulfated peak 8 (gray arrow) and an increase in monosulfated peak 5 (white arrow). (C) The EIC
of the dp2s after chemical 2-O-desulfation is shown in the dotted lines. The disulfated peak 8 as well as the monosulfated peak 5 decreased (gray
arrows). Peaks 2 and 7 increased after desulfation (white arrows). (D) The EIC of the dp2s after chemical 6-O-desulfation is shown in the dotted
lines. Disulfated peaks 8 and 9 and monosulfated peaks 3 and 4 decreased after desulfation (gray arrows). Unsulfated peaks 1 and 2 and
monosulfated peak 5 increased after removal of 6-O-sulfate (white arrows).
disaccharide peak was collected and subjected to tandem mass
spectrometry in addition to chemical O-desulfation and
uronidase degradation to determine the uronic acid epimeriza-
tion and the sulfate position. EDD and EID were performed on
each of the isomeric disaccharides. The two MS/MS techniques
provided unique product ion spectra of each of the isomers,
useful for identification of the position of the sulfate groups
(Supporting Information Figures S-7 and S-8). The rms mass
accuracy of the product ion assignments averaged 0.5 ppm. The
EDD mass spectra are consistent with the assignment of 2-O
and 6-O-sulfation sites of the monosulfated dp2s.
residue, either at the C3 or C6 position, and can have the
structures shown in Supporting Information Figure S-5F,G,H.
The MS2 spectra also showed several product ions resulting
from a cross-ring cleavage: 2,5X1-CO2H (m/z 256), 1,5X1 (m/z
271), 0,2X1 (m/z 285), 0,3X1-2H (m/z 313), and 0,3X1 (m/z
315).
Most of the observed glycosidic bond and cross-ring cleavage
product ions were common to all tandem mass spectra but with
different abundances. A few peaks were not common to all
spectra. The product ions 1,3X0 and Y1−CH2O are observed
only in the mass spectra of peaks 6 and 7 but are significantly
more intense in the spectrum of peak 6. The presence of 1,3X0
in the EDD of peaks 6 and 7 is consistent with results from
previous sections showing that these two peaks are 6-O-
sulfated. A product ion at m/z 341, which is likely the loss of
H2O from 1,4A2 or 1,3X1 or 2,4X1, is observed only in the EDD
spectrum of peak 7 and not in peak 6. In addition, the product
ions 0,3X1-2H (m/z 313) and 2,4A2/1,4X1/3,5X1-2H (m/z 329)
are more intense in the EDD and EID spectra of peak 7
compared to peak 6. These peaks can, thus, distinguish the 6-
O-sulfated peaks 6 (Supporting Information Figure S-7F) and 7
(Supporting Information Figure S-7G) which differ only in
their hexuronic acid. For peak 5 (Supporting Information
Figures S-5C and S-6C), the EID mass spectrum showed five
diagnostic fragment ions. Three product ions result from
glycosidic cleavage wherein the charge is retained on the uronic
acid containing the sulfate group: B1-CO2 (m/z 211), B1 (m/z
Some product ions in the EDD of [M − 2H]2‑ and EID of
[M − H]− (Supporting Information Figures S-7 and S-8) were
common to all isomers and were generated from the neutral
loss from the precursor ion. An abundant neutral loss product
ion was m/z 357, assigned as [M − CO2H − H2O]−. In the
EID, the abundant product ions m/z 339 and 388 likely
correspond to a loss of SO3 and CH3O, respectively. For all
isomers, both EDD and EID resulted in the presence of cross-
ring cleavages in addition to glycosidic cleavages. For peaks 3, 4,
6, and 7 (Supporting Information Figures S-5A,B,D,E and S-
6A,B,D,E), the presence of product ions at m/z 225 and 243
corresponds to glycosidic bond cleavages where charge is
retained on the reducing end portion of the molecule
containing the sulfate group, to form Z1 and Y1 ions,
respectively. The glycosidic bond cleavage product ions
establish that the site of sulfation lies within the aManR
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Table 1. Disaccharides Obtained by Nitrous Acid Depolymerization and PGC and the Effect of Exoglycosidases and Chemical-
O-Desulfation
peak
disaccharide
GlcA-aManR
β-glucuronidase
α-iduronidase
2-O-desulfation
6-O-desulfation
1
2
3
4
5
6
7
8
9
b
IdoA-aManR
↓ (70%)
c
GlcA-aMan(3S)R
GlcA-aMan(3S)/(6S)R
IdoA(2S)-aManR
GlcA-aMan(6S)R
IdoA-aMan(6S)R
IdoA(2S)-aMan(6S)R
GlcA-aMan(3S)(6S)R
0
0
↓ (50%)
c
↓ (90%)
c
c
↓ (60%)
↓ (95%)
a
c
↓ (45%)
0
0
↓ (100%)
a
c
↓ (65%)
↓ (100%)
d
d
↓ (95%)
↓ (95%)
d
0
↓ (95%)
a
b
Value is an average of the decrease in abundance of dp2s from HSBK and HSPIM. Value is based on the decrease of abundance of dp2 from
c
d
heparosan. Value is based on the decrease of abundance of purified monosulfated dp2s. Value is an average of the decrease in abundance of dp2s
from HSBK, HSPIM, and heparin.
255), and C1 + H (m/z 272). The other two product ions,
1,4A1-2H (m/z 167) and 1,5A1 (m/z 227), result from cross-ring
cleavages. The sulfate group of peak 5 is assigned to the C2
position of the uronic acid as shown in Supporting Information
Figure S-5I, consistent with the results from the previous
sections.
corresponding increase in IdoA-aManR, indicating that this
peak is IdoA(2S)-aManR. Also, treatment with MTSTFA
resulted in removal of a small (∼5%) amount of the 2-O-
sulfate. Both peaks 6 (Supporting Information FigureS-8D) and
7 (Supporting Information FigureS-8E) were unaffected by the
treatment with alkali but disappeared after 6-O-desulfation. For
peak 6, there was a corresponding increase in peak 1 (GlcA-
aManR) suggesting that this peak is GlcA-aMan(6S)R. On the
other hand, there was an increase in peak 2 (IdoA-aManR) after
removal of the 6-O-sulfate from peak 7 indicating that this peak
is IdoA-aMan(6S)R. These results are in agreement with those
discussed in previous sections.
Each monosulfated disaccharide was subjected to chemical
O-desulfation to determine the uronic acid epimerization. After
the chemical treatment, an increase in either GlcA-aManR or
IdoA-aManR was expected, resulting from the removal of the
sulfate. The results are shown in Supporting Information Figure
S-8. In the figure, the dotted trace represents the EIC of m/z
339 of epimerized and N-sulfated K5 polysaccharide. In a
previous section, it was demonstrated that peaks 1 and 2
correspond to GlcA-aManR and IdoA-aManR, respectively. For
each series, the upper panel represents the EIC of m/z 419 for
the purified monosulfated dp2, the middle panel shows the
effect of 2-O-desulfation, and the lower panel is the effect of 6-
O-desulfation. For peak 3 (Supporting Information FigureS-
7A), 2-O-desulfation had no effect. After 6-O-desulfation, there
was a ∼50% decrease in peak 3 and a corresponding increase in
GlcA-aManR, indicating that this peak is GlcA-aMan(3S)R or
GlcA-aMan(6S)R. Similarly, 6-O-desulfation resulted in a
decrease in the intensity of peak 4 (Supporting Information
FigureS-7B) and an increase in GlcA-aManR, suggesting that
this peak is also either GlcA-aMan(3S)R or GlcA-aMan(6S)R.
However, the removal of sulfate for peak 4 was greater (∼90%)
compared to peak 3 which is consistent with the conclusion
that these peaks represent different structures. In fact, the effect
of 6-O-desulfation was least in peak 3 (Supporting Information
FigureS-7A) compared to peaks 4 (Supporting Information
FigureS-7B), 6 (Supporting Information FigureS-7D), and 7
(Supporting Information FigureS-7E), which suggests that this
peak has a different sulfate position, a 3-O-sulfate instead of a 6-
O-sulfate. This conclusion is supported by the fact that peak 3
is abundant for heparin and HSPIM but has very low
abundance for HSBK (Figure 1). In addition, peak 3 is of
very low abundance in HS from aorta, lung, and the low salt
fraction of kidney and is significant in abundance only for
intestine and the high salt fraction of kidney (Supporting
Information Figure S-4). This pattern is consistent with peak 3
corresponding to GlcA-AMan3SR since its abundance is highest
for tissues known to have high anticoagulant activity. The
anticoagulant activity requires a 3-O-sulfation.48,49
Using the exoglycosidases, chemical O-desulfation, and
tandem mass spectrometry, the elution order in PGC of the
HONO-derived dp2s has been determined (Table 1).
CONCLUSIONS
■
A method for disaccharide analysis of HS and heparin samples
using deaminative cleavage by HONO in conjunction with
PGC-MS has been described in this work. Results demonstrate
that PGC is suitable for the separation of HONO-derived dp2s
with different sulfate groups. In addition, PGC allows
separation of uronic acid epimers and isomeric glycans prior
to MS and tandem MS. Another advantage of PGC is that the
mobile phase used makes it well suited for interface with mass
spectrometry. Lastly, the disaccharide composition obtained
from a combination of HONO treatment and PGC-MS is
comparable to that of enzymatic digestion and SEC-MS. This
analytical method provides data on uronic acid epimers that is
essential for modeling of HS structure based on compositional
analysis.22
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional information as described in the text. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Address: Department of Biochemistry, Boston University
School of Medicine, 670 Albany St., Rm. 509, Boston, MA
02118. E-mail: jzaia@bu.edu. Phone: 617-638-6762. Fax: 617-
638-6761.
Notes
For peak 5 (Supporting Information Figure S-8C), there was
∼60% decrease in intensity after 2-O-desulfation and a
The authors declare no competing financial interest.
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dx.doi.org/10.1021/ac3016054 | Anal. Chem. 2012, 84, 7539−7546

Analytical Chemistry
Article
(36) Staples, G. O.; Shi, X.; Zaia, J. J. Biol. Chem. 2010, 285, 18336−
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ACKNOWLEDGMENTS
■
Support for this research was provided by NIH grants
P41RR10888, P41GM104603, R01HL098950, and
S10RR025082.
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