Glycobiology vol. 16 no. 6 pp. 502–513, 2006
doi:10.1093/glycob/cwj093
Advance Access publication on February 20, 2006
A tandem mass spectrometric approach to determination of chondroitin/dermatan sulfate
oligosaccharide glycoforms
May Joy C. Miller2, Catherine E. Costello2,
Anders Malmström3, and Joseph Zaia1,2
2
Department of Biochemistry, Boston University School of Medicine,
715 Albany Street, R-806, Boston, MA 02118; and 3Department of Cell and
Molecular Biology, Lund University, BMC B11, S-221 84, Lund, Sweden
Received on October 27, 2005; revised on January 26, 2006; accepted on
February 15, 2006
Dermatan sulfate (DS) chains are variants of chondroitin sulfate (CS) that are expressed in mammalian extracellular
matrices and are particularly prevalent in skin. DS has been
implicated in varied biological processes including wound
repair, infection, cardiovascular disease, tumorigenesis, and
fibrosis. The biological activities of DS have been attributed
to its high content of IdoA(a1–3)GalNAc4S(b1–4) disaccharide units. Mature CS/DS chains consist of blocks with high
and low GlcA/IdoA ratios, and sulfation may occur at the
4- and/or 6-position of GalNAc and 2-position of IdoA.
Traditional methods for the analysis of CS/DS chains involve
differential digestion with specific chondroitinases followed
by steps of chromatographic isolation of the products and disaccharide analysis on the individual fraction. This work
reports the use of tandem mass spectrometry to determine the
patterns of sulfation and epimerization of CS/DS oligosaccharides in a single step. The approach is first validated and
then applied to a series of skin DS samples and to decorins
from three different tissues. DS samples ranged from 74 to
99% of CSB-like repeats, using this approach. Decorin samples ranged from 30% CSB-like repeats for those samples
from articular cartilage to 75% for those from sclera. These
values agree with known levels of glucuronyl C5-epimerase in
these tissues.
Key words: chondroitin sulfate/decorin/dermatan sulfate/
glycosaminoglycan/mass spectrometry
Introduction
Glycosaminoglycan (GAG) glycoforms are of particular
interest because of their varied biological properties. Proteoglycans (PGs)/GAGs are predominantly found on cellular membranes and in the extracellular matrix of
mammalian tissues. They interact with numerous proteins,
growth factors, and cell surface receptors and thus serve a
variety of important roles in cell proliferation, differentiation, adhesion, migration, and recruitment of neutrophils
(Hocking et al., 1998; Bernfield et al., 1999; Trowbridge
1
To whom correspondence should be addressed; e-mail: [email protected]
and Gallo, 2002). GAGs are linear, sulfated polysaccharides that are attached by a linker to a serine residue of the
core protein of a proteoglycan; the structures of GAGs
bound to a given core protein are known to vary depending
on the environment (tissue, developmental disease, or
cytokine state) (Faissner et al., 1994; Kitagawa et al., 1997;
Clement et al., 1998; Tiedemann et al., 2005). The dynamic
nature and biological importance of GAG glycoforms drive
the need for a rapid and sensitive means of determination
of their sulfation and epimerization patterns. Dermatan
sulfate (DS) is a chondroitin sulfate (CS) variant that exhibits anticoagulant activity in both animals and humans and
has been incorporated into antithrombic therapies (Maimone
and Tollefsen, 1990; Cofrancesco et al., 1994; Di Carlo et al.,
1999). In contrast to other antithrombic GAGs, DS is effective on both free and fibrin-bound thrombin (Bendayan
et al., 1994; Liaw et al., 2001) and has low hemorrhagic
complications (Cofrancesco et al., 1994; Di Carlo et al.,
1999). Scleral proteoglycans interact via their DS chains,
demonstrating the multifaceted potentials of GAGs in promoting multifaceted binding with other macromolecules
(Fransson et al., 1982).
DS interactions with fibroblast growth factors FGF-2
(Penc et al., 1998) and FGF-7 (Trowbridge et al., 2002)
have been shown with respect to cellular proliferation and
wound repair, respectively. Interactions with hepatic
growth factor/scatter factor establish that DS plays roles in
tumorigenesis and metastasis (Lyon et al., 2002). Oversulfated DS chains play roles in promoting neurite outgrowth
in embryonic brain (Hikino et al., 2003). Decorin, a DScontaining proteoglycan predominant in skin, acts as an
inflammation regulator through interaction with C1q
(Krumdieck et al., 1992) and transforming growth factorbeta (Yamaguchi et al., 1990) and may play an important
role in the inflammatory process of Lyme disease (Guo
et al., 1995).
Although the IdoA(α1–3)GalNAc4S(β1–4) disaccharide unit is the most highly expressed in mammalian DS,
analyses of several preparations reveal heterogeneity
within the polysaccharide chain (Poblacion and Michelacci,
1986). This is due to the biosynthetic modifying reactions
that generate DS from nascent chondroitin chains of composition [GlcA(β1–3)GalNAc(β1–4)]n after the initial
polymerization; epimerization of GlcA to IdoA occurs
before sulfation (Malmstrom and Aberg, 1982; Malmstrom, 1984). Mammalian DS consists of domains of high
percentage of IdoA(α1–3)GalNAc4S(β1–4) and those
with high GlcA(β1–3)GalNAc4S(β1–4). Variable quantities of GlcA(β1–3)GalNAc6S(β1–4) are also present
(Poblacion and Michelacci, 1986). DS chains from the
body of an ascidian Ascidia nigra have a repeat unit of
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502
MS of CS/DS
IdoA2S(α1–3)GalNAc6S(β1–4) (Pavao et al., 1995) and
lack the anticoagulant activity of 4-sulfated DS (Vicente
et al., 2001). Monoclonal antibodies generated against
Ascidia DS have demonstrated the presence of
IdoA2S(α1–3)GalNAc6S(β1–4) repeats in mouse brain
(Bao et al., 2005).
Because the ability of GAGs to bind protein receptors
largely depends on their structure, the number of disaccharide repeats (size), and sulfation pattern, analytical techniques to determine GAG fine structure are highly
valuable. Total uronic acid content for GAG preparations
may be determined using the carbazole reaction (Meyer and
Rapport, 1950; Rapport et al., 1951; Bitter and Muir,
1962). Chemical measurement of IdoA and GlcA content
entails reaction of uronate carboxyl groups with a carbodiimide, followed by reduction and acid hydrolysis. The
monosaccharide products, differing for GlcA and IdoA, are
then per-O-benzoylated, separated, and quantified using
chromatography (Karamanos et al., 1988). The IdoA content of CS/DS chains may be determined by differential
chondroitinase digestion followed by chromatographic separation (Coster et al., 1975). The differential chondroitinase
digestion approach has been used in conjunction with subsequent size-exclusion chromatography (Malmstrom and
Fransson, 1975), reversed-phase ion pairing high-performance liquid chromatography (RPIP-HPLC) (Karamanos
et al., 1988), or slab gel electrophoresis (Fransson et al.,
1990; Cheng et al., 1994). Oligosaccharide products of
chondroitinase reactions isolated by RPIP-HPLC or other
techniques may be subjected to additional chemical or
enzymatic degradative steps (Theocharis et al., 2001) followed by monosaccharide or disaccharide analysis. Highly
sensitive chromatographic (Toyoda et al., 1999, 2000), capillary electrophoretic (Karamanos et al., 1995; Mitropoulou
et al., 2001; Lamari et al., 2002), fluorescence-assisted carbohydrate electrophoresis (Calabro, Benavides, et al., 2000;
Calabro, Hascall, et al., 2000), and mass spectrometric
(Desaire and Leary, 2000) methods are available for CS/DS
disaccharide analysis. To determine uronic acid epimers
from disaccharide analysis, one may use a chemical degradation method. The intact GAG chains are deacetylated by
hydrazinolysis and subjected to deaminative cleavage using
nitrous acid at pH 4. The resulting disaccharides, each containing dehydromannose at the reducing end and an
unmodified uronic acid residue, may be reduced with
sodium borotritide or tagged with a chromophore or fluorophore before chromographic or electrophoretic separation (Guo and Conrad, 1989).
Measurement of the mass of a CS/DS oligosaccharide
determines the composition with respect to the number of
uronic acid, N-acetylhexosamine, and sulfate units (Carr
and Reinhold, 1984; Takagaki et al., 1992; Sugahara et al.,
1994). Tandem mass spectrometry (MS) determines the
number of sulfate groups per disaccharide unit and provides a measure of IdoA(α1–3)GalNAc4S(β1–4) content in
the target oligosaccharide (Zaia et al., 2001, 2003) based on
product ion abundances.
We have used a liquid chromatography (LC)–MS platform for determining CS/DS glycoform distribution
(Hitchcock et al., 2006). The present work describes the
principles behind this approach and extends the analysis to
longer oligosaccharides. A series of dermal DS samples isolated under different conditions are analyzed, in addition to
a series of decorins from different tissues. The CS/DS
chains are partially depolymerized using chondroitinase
ABC and the resultant oligosaccharides analyzed using tandem MS. This approach is used to compare the expression
of oligosaccharide glycoforms among the different samples.
Such comparisons would be significantly more laborious
using traditional techniques. These experiments demonstrate the principle and validity of a new method for glycoform analysis of CS/DS chains, one that will be useful in the
emerging field of glycomics.
Results
Partial depolymerization of CS/DS samples
Oligosaccharides were generated from CS/DS samples by
stopping chondroitinase ABC digestions at a time point
found to produce 232 nm absorbance of 30% of its maximum value (see Materials and methods for details). The oligosaccharides were fractionated using high-performance
size-exclusion chromatography with 232 nm detection.
Oligosaccharide length will be given as degree of polymerization (dp) and the number of monosaccharide units. Peak
area percentages for dp2, dp4, and dp6 for each of the samples are summarized in Table I. The size-exclusion chromatograms displayed no defined peaks greater than dp6,
and the 232-nm baseline was elevated before the elution of
this oligomer. Thus, the reported values include the area of
this baseline elevation, listed as “>dp6.”
MS of CS/DS oligosaccharides
The compositions of oligosaccharides with a 4,5-unsaturated
uronic acid residue at the nonreducing end will be given
preceded with the Δ symbol. Figure 1 is a representative
electrospray (ESI) mass spectrum of size-exclusion chromatography fractions corresponding to dp4 (A) and dp6 (B).
The ion at m/z 458 corresponds to [M-2H]2– for Δdp4 (A)
Table I. Relative chromatographic peak areas for CS/DS samples
partially digested with chondroitinase ABC
dp2
dp4
dp6
>dp6
CSA
48.35
9.16
9.98
32.5
CSB
38.92
18.27
18.53
24.3
CSC
55.30
19.19
9.48
16.0
CS6
58.02
21.38
9.13
11.5
DS18
35.80
14.48
16.59
33.1
DS36
37.93
10.91
11.53
39.6
DS50
43.13
15.24
12.71
28.9
ACD
39.80
15.72
—
44.5
CD
55.16
29.19
—
15.7
SD
42.69
23.03
—
34.3
Chondroitinase ABC digestions were stopped at 30% completion, and
separated using high-performance size-exclusion chromatography (SEC),
as described in the Materials and methods section. The relative peak areas
for Δ-unsaturated dp2, dp4, and dp6 oligosaccharides are given.
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M.J.C. Miller et al.
Fig. 1. Representative ESI mass spectra of partially depolymerized and
purified CS/DS size-exclusion chromatography fractions in the negative
mode, (A) the spectrum obtained from the dp4 fraction and (B) that from
the dp6 fraction.
and [M-3H]3– for Δdp6 (B). Because this ion is isobaric
for Δ-unsaturated CS/DS oligosaccharides of composition
Δ(HexA)n(GalNAc)n(SO3)n, careful isolation of the individual oligomers was performed using high-performance
size-exclusion chromatography. The isotope pattern of the
ion at m/z 458 (Figure 1A, inset) shows peaks separated by
0.50 μ that signify a charge state of 2–, the neutral mass of
which (918.10 Da; Table II) matches a composition of
Δ(HexA)2(GalNAc)2(SO3)2. The inset in Figure 1B shows
peaks separated by 0.33 μ, signifying a charge state of 3–,
the neutral mass of which (1377.24 Da; Table II) is consistent with a composition of Δ(HexA)3(GalNAc)3(SO3)3. The
absence of an ion at m/z 458.38 from Figure 1A shows that
the dp4 fraction is not contaminated with dp6. The absence
of an ion at m/z 458.55 from Figure 1B shows that there is
no dp4 in the dp6 fraction. These negative electrospray ionization patterns produced from CS/DS oligosaccharides are
consistent with the previous reports (Desaire and Leary,
2000; Zaia and Costello, 2001; Zaia et al., 2001, 2003; Chai
et al., 2002; Zamfir et al., 2003).
Assignments for all ions observed in the mass spectra of
CS/DS dp4 and dp6 fractions are listed in Table II.
The ions at m/z 498 in Figure 1A correspond to Δ(HexA)2
(GalNAc)2(SO3)3 and m/z 484 in Figure 1B to Δ(HexA)3
(GalNAc)3(SO3)4 (Table II). These CS/DS oligosaccharides
contain more than one sulfate group per disaccharide
repeat and are termed oversulfated. In addition, an [M-2H]2–
ion corresponding to a saturated oligosaccharide of composition of (HexA)3(GalNAc)3(SO3)3 was detected at m/z
696.5 in Figure 1B. This composition matches that expected
for an oligosaccharide derived from the nonreducing terminus of the intact CS/DS chain, by virtue of the fact that the
neutral mass is consistent with a saturated structure. The
ion at m/z 464 in Figure 1B corresponds to [M-3H]3– for the
same composition. The ion at m/z 899 in Figure 1B corresponds to the composition [Δ(HexA)2(GalNAc)2(SO3)2–
H2O] (Table II). Gentle ionization conditions were used,
and no losses of SO3 or other signs of in-source fragmentation were observed in Figure 1. It is therefore likely that the
species giving rise to m/z 899 exists in solution and is not an
artifact of the MS measurement. Although the oligosaccharide composition may be determined from the molecular
weight information obtained from the ESI mass spectra,
Table II. Mass spectral ion assignments for CS/DS Δdp4 and Δdp6 oligosaccharides
dp4
dp6
m/z
Ion
Description
Composition
305.06
[M-3H]3–
Δ(HexA)2(HexNAc)2(SO3)2
C28H41N2O28S2
918.13
918.21
458.05
[M-2H]2–
Δ(HexA)2(HexNAc)2(SO3)2
C28H40N2O28S2
918.13
918.14
498.03
[M-2H]2–
Δ(HexA)2(HexNAc)2(SO3)3
C28H40N2O31S3
998.07
998.08
917.01
[M-H]–
Δ(HexA)2(HexNAc)2(SO3)2
C28H41N2O28S2
918.13
918.02
4–
Δ(HexA)3(HexNAc)3(SO3)3
C42H59N3O42S3
1377.20
1377.28
3–
343.31
[M-4H]
Observed mass
458.07
[M-3H]
Δ(HexA)3(HexNAc)3(SO3)3
C42H60N3O42S3
1377.20
1377.24
464.08
[M-3H]3–
(HexA)3(HexNAc)3(SO3)3
C42H62N3O43S3
1395.21
1395.27
484.71
[M-3H]3–
Δ(HexA)3(HexNAc)3(SO3)4
C42H60N3O45S4
1457.16
1457.16
687.55
[M-2H]2–
Δ(HexA)3(HexNAc)3(SO3)3
C42H61N3O42S3
1377.20
1377.12
696.51
2–
[M-2H]
(HexA)3(HexNAc)3(SO3)3
C42H63N3O43S3
1395.21
1395.27
727.52
[M-2H]2–
Δ(HexA)3(HexNAc)3(SO3)4
C42H61N3O45S4
1457.16
1457.06
899.01
[M-H]–
Δ(HexA)2(HexNAc)2(SO3)2-H2O
C28H40N2O27S2
900.12
900.01
The Δ symbol is used to signify oligosaccharides with a 4,5-unsaturated uronic acid at the nonreducing terminus.
504
Calculated mass
MS of CS/DS
detailed structural information such as sulfation positions
and epimerization state is not produced.
Tandem MS of CS/DS Ddp4 glycoforms
To determine positions of sulfation and epimerization, we
compared abundances of the fragment ions formed from
DS oligosaccharides with those produced from commercial
CS/DS standards with known epimerization and GalNAc
sulfate positions. It was established previously that tandem
MS product ion abundances of CS oligosaccharides reflect
sulfation position at GalNAc residues (Zaia et al., 2001;
McClellan et al., 2002) and epimerization of HexA residues
(Zaia et al., 2003). Thus, three commercial standards were
used, each of which is ∼90% pure. These were CSA
(GlcA(β1–3)GalNAc4S(β1–4))n, CSB (IdoA(α1–3)Gal
NAc4S(β1–4))n, and CSC (GlcA(β1–3)GalNAc6S(β1–4))n.
Furthermore, to focus the analysis on a single uronic acid
that is unaltered by lyase digestion, we first investigated
Δdp4 series.
Figure 2 shows the tandem mass spectra of Δdp4 oligosaccharides generated from CS standard preparations,
CSA (Figure 2A), CSB (Figure 2B), and CSC (Figure 2C).
The doubly charged precursor ion is typically present as the
most abundant charge state, and product ions resulting
from glycosidic bond cleavages are abundant, whereas
those from losses of SO3 are in low abundance. Although
the three CS/DS Δdp4 isomers dissociate to form ions with
identical m/z values, differences in abundances of the fragment ions characterize each other. Specifically, there are six
signature ions, the abundances of which differentiate the
three isomers. The Δdp4 oligosaccharides derived from
CSA are characterized by high abundance of Y11– (m/z 300)
and B31– (m/z 616) ions relative to those observed for the
other two isomers (Figure 2A). CSB Δdp4 oligosaccharides
are characterized by the high abundance of 0,2X32– (m/z
400) and Y32– (m/z 379) ions relative to the other two isomers. Abundant [M-SO3]2– (m/z 418) and C32– (m/z 316)
ions characterize CSC Δdp4 oligosaccharides. These fragment ions are summarized in Table III. In low-energy collision-induced dissociation (CID) experiments, product ion
abundances reflect the lability of the covalent bonds that
are cleaved during the tandem MS dissociation. Thus, the
sulfation and epimerization positions influence the lability
of certain bonds in the oligosaccharide ions that are
reflected by the observed ion abundances.
The Y11– ion contains the sulfate group on the reducing
terminal GalNAc residue of the precursor oligosaccharide
ion. Abundance of this ion correlates directly with the presence of an oligosaccharide with a CSA-like GlcA(β1–3)
GalNAc4S(β1–4) repeat. Similarly, the B31– ion, which
contains the internal sulfated GalNAc residue, is produced
by cleavage of the same glycosidic bond, and its abundance
correlates with the presence of the same repeat. It is likely
that the abundance of both the Y11– and the B31– ions
reflect the position of sulfation on the reducing end
GlcA(β1–3)GalNAc4S disaccharide. The [M-2H]2– ion of
Δdp4 derived from CSC dissociates to produce abundant
[M-SO3]2– and C32– ions. The C32– ion contains the internal
sulfated GalNAc residue, but the ion abundance of the glycosidic bond cleavage could be influenced by the sulfate
Fig. 2. Tandem mass spectra of Δ-unsaturated dp4 derived from CSA
(A), CSB (B), and CSC (C). The structure of Δ-unsaturated dp4 from CSA
is shown in (D) with product ion assignments.
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M.J.C. Miller et al.
Table III. Tandem mass spectrometric signature ions for CS/DS Δdp4
and Δdp6 oligosaccharides
Ion
Δdp4 [M-2H]2–
Table IV. Percent total ion abundances for signature ions for Δdp4
derived from CSA, CSB, and CSC
Composition
Calculated m/z Observed m/z
Ion
CSA
CSB
CSC
C28H40N2O28S2
458.06
Y11–
33.91 ± 0.61
10.14 ± 0.56
19.88 ± 0.48
1–
458.05
Y1
C8H14NO9S
300.04
300.06
B3
11.22 ± 0.33
2.92 ± 0.73
7.74 ± 0.52
B31–
C20H26NO19S
616.08
616.04
Y32–
1.63 ± 0.27
3.18 ± 0.15
0.68 ± 0.05
0,2X 2–
3
Y32–
C32–
C24H36O24N2S2
400.05
400.06
0.66 ± 0.05
2.41 ± 0.31
0.44 ± 0.04
379.06
0,2X 2–
3
C32–
0.96 ± 0.03
0.78 ± 0.08
2.06 ± 0.18
0.89 ± 0.09
0.72 ± 0.64
2.23 ± 0.05
1–
C22H35O23N2S2
316.54
316.57
[M-SO3]2–
418.08
418.09
The data are plotted as a bar graph in Figure 3.
C42H60N3O42S3
458.06
458.05
C20H27O20NS
[M-SO3-2H]2– C28H40O25N2S
Δdp6 [M-3H]3–
Y11–
379.05
C8H14NO9S
300.04
300.05
2–
C34H46N2O33S2
537.07
537.06
X53–
C38H56N3O38S3
419.39
419.39
Y53–
C36H54N3O37S3
405.38
405.39
C52–
C34H48N2O34S2
546.08
546.03
C42H60N3O39S2
431.41
431.41
B5
0,2
3–
[M-SO3-3H]
position of the reducing end GlcA(β1–3)GalNAc6S disaccharide during the CID process. Loss of sulfate, [M-SO3]2–,
is nonspecific as to the originating GlcA(β1–3)GalNAc6S
disaccharide but is indicative of CSC-like structures. The
0,2X 2– and Y 2– ion abundances are enhanced when CSB3
3
like IdoA(α1–3)GalNAc4S(β1–4) repeats are present.
Standards for Δdp4 and Δdp6 oligosaccharides containing mixed GalNAc 4- and 6-sulfation patterns are not available. However, for Δdp4, the abundances of the CSA
signature ions, Y11– and B31–, reflect the position of sulfation of the reducing end GalNAc residue. It is therefore
likely that all tetrasaccharides with GalNAc-4-sulfate on
the reducing end will produce abundant CSA-like signature ions. CSC-like signature ions correspond to C32– and
[M-SO3]2–. The abundance of the C32– ion varies according
to the position of sulfation of the reducing terminal GalNAc residue and that of the [M-SO3]2– ion varies according
to the presence of GalNAc-6-sulfate at either position in the
tetramer. Thus, it would be expected that a mixed tetramer
with GalNAc-6-sulfate on the reducing end would produce
an abundant C32– ion, but that of the [M-SO3]2– ion would
be intermediate.
Signature ion patterns
The use of signature ions to distinguish the three CS isomeric oligosaccharides can best be seen when their percent
total ion abundances (Table IV) are plotted. The Y11– and
B31– ions are most abundant, expressed as a percent of the
sum of all product ion abundances (Figure 3A), for CSA
Δdp4. The Y32– and 0,2X32– ions are most abundant for
Δdp4 derived from CSB. The C32– and [M-SO3]2– ions are
most abundant for Δdp4 derived from CSC. The data are
replotted in Figure 3B to show how each CS type produces
a characteristic pattern of these diagnostic fragment ions.
Thus, a given CS sample produces a pattern that is a linear
combination of CSA-, CSB-, and CSC-like signature ions.
506
Fig. 3. Signature ion patterns for Δ-unsaturated dp4 derived from
standard preparations of CSA, CSB, and CSC. The abundances of
selected product ions are compared in (A). The data are replotted in
(B) to show how the ion abundances vary according to the type of CS
from which the Δ-unsaturated dp4 was derived.
These ions will be used to characterize oligosaccharides
derived from CS/DS samples of biological interest, as discussed in the Analysis of varied DS samples and decorin
section.
MS of CS/DS
Fig. 5. Signature ion patterns for Δ-unsaturated dp6 derived from standard preparations of CSA, CSB, and CSC. The abundances of selected
product ions are compared in (A). The data are replotted in (B) to show
how the ion abundances vary according to the type of CS from which the
Δ-unsaturated dp6 was derived.
Fig. 4. Tandem mass spectra of the Δ-unsaturated dp6 derived from standard preparations of CSA (A), CSB (B), and CSC (C). The fragment ion
assignments are shown in (D) for Δ-unsaturated dp6 derived from CSA.
and B52– (m/z 537) ions are highest for CSA-derived Δdp6
oligosaccharides (Figure 4A). Although these ions are
abundant for Δdp6 oligosaccharides derived from CSC, the
abundance as a percent of the total is higher for Δdp6
derived from CSA because of the lower abundances of
other product ions. The 0,2X53– (m/z 419) and Y53– (m/z
405) ions have the highest percent total ion abundances for
Δdp6 derived from CSB (Figure 4B), whereas [M-SO3]3–
(m/z 431) and C52– (m/z 546) ions are observed in greatest
abundance for those from CSC (Figure 4C). Figure 5A
shows how the abundances of the signature ions vary for
different types of CS. The data are replotted in Figure 5B to
show how the different forms of CS Δdp6 produce characteristic ion patterns.
Tandem MS of CS/DS Ddp6 glycoforms
As with the CS/DS Δdp4, signature ions characterize the
Δdp6 oligosaccharides derived from standard CSA, CSB,
and CSC. A summary of these ions is provided in Table III.
The percent total ion abundances for the Y11– (m/z 300)
Analysis of varied DS samples and decorin
To demonstrate the utility of this method, we analyzed several CS/DS samples, the Δdp4 signature ion profiles of
which are shown in Figure 6. The CS6 sample was purified
507
M.J.C. Miller et al.
Fig. 6. Signature ion profile for Δ-unsaturated dp4 oligosaccharides
derived from CS and DS samples. CS6 is from horse nasal septum.
DS18, DS36, and DS50 are from pigskin.
from horse nasal septum and several DS preparations,
namely DS18, DS36, and DS50 were purified from pigskin.
From the signature ion patterns, it can easily be confirmed
that Δdp4 oligosaccharides derived from DS18, DS36, and
DS50 display signature ion patterns similar to those of
standard CSB. This indicates that a high percentage of the
Δdp4 oligosaccharides in each of these samples contain the
IdoA(α1–3)GalNAc4S(β1–4) sequence. Those from CS6,
by contrast, display signature ion patterns consistent with a
high percentage of GlcA(β1–3)GalNAc6S(β1–4) repeats.
Although the signature ion patterns of these samples qualitatively resemble those of the standards, the heights of the
bars differ. For instance, samples DS18, DS36, and DS50
show an apparent increase in the height of CSA-like ions,
suggesting varying GlcA(β1–3)GalNAc4S(β1–4) content of
DS18, DS36, and DS50. Also, the middle bar, which represents CSB-like ions, decreases from DS18 to DS50, which
reflects the decreasing IdoA(α1–3)GalNAc4S(β1–4) content in the DS samples. However, the CSC-like signature
ions are observed consistently in low abundances among
the DS samples, indicating a minimal content of GlcA(β1–3)
GalNAc6S(β1–4)repeats. CS6 Δdp4 oligosaccharides have
significantly more abundant CSC-like signature ions than
do those derived from the commercial CSC standard, suggesting that they contain a higher percentage of GlcA(β1–3)
GalNAc6S(β1–4) repeats.
The signature ion pattern for the decorin samples is
shown in Figure 7. All three decorins tested—bovine articular cartilage decorin (ACD), human cervix decorin (CD),
and bovine sclera decorin (SD)—exhibit a CSB-like signature ion pattern, consistent with a high content of
IdoA(α1–3)GalNAc4S(β1–4) in the Δdp4 oligosaccharides
generated therefrom. A decreasing CSA-like signature ion
contribution is indicated, proceeding from ACD to CD to
SD, which again suggests that the content of GlcA(β1–3)
GalNAc4S(β1–4) sequences varies among Δdp4 generated
from the three decorins. Moreover, the CSB-like ions
increase from ACD to CD to SD, suggesting that Δdp4
508
Fig. 7. Signature ion profile for Δ-unsaturated dp4 oligosaccharides
derived from decorin samples: articular cartilage decorin (ACD),
human cervix decorin (CD), and bovine sclera decorin (SD).
generated from SD has the greatest IdoA(α1–3)
GalNAc4S(β1–4) content of the three decorins. However,
the CSC-like ions have the greatest contribution for ACD
compared with almost insignificant contribution for CD
and SD, suggesting that ACD contains the greatest
GlcA(β1–3)GalNAc6S(β1–4) percentage among the three
decorins. For the Δdp6 series, the signature ion profile of
DS samples and decorin is similar to that of the Δdp4 series.
Quantitative analysis of each isomeric contribution
As was pointed out earlier, the signature ion abundances
vary directly with the abundances of CSA-, CSB-, and
CSC-like disaccharide repeats in each sample. Because the
signature ion abundances for a given sample are a linear
combination of the three repeat types, the percentages of
CSA-, CSB-, and CSC-like ions required to produce the
observed patterns can be calculated. Tandem MS ion abundances have been used to quantitate CS and heparin disaccharides (Desaire and Leary, 2000; Saad and Leary, 2003).
Using similar principles applied to the Δdp4 oligosaccharide signature ion abundances, the following equations
were applied to calculate the percent contribution of CSA,
CSB, and CSC:
AY1 + BY1 + CY1 = DY1 ,
AY3 + BY3 + CY3 = DY3 ,
and
A(M-SO3 ) + B(M-SO3 ) + C(M-SO3 ) = D(M-SO3 )
where Y1, Y3, and M-SO3 are the signature ions for CSA-,
CSB-, and CSC-like, respectively; A, B, and C are the
unknown contributions of CSA, CSB, and CSC isomers;
and D is the contribution from the unknown sample. The
percent total ion abundance of each signature ion from the
standards is substituted on the left side of the equations and
MS of CS/DS
those from the sample on the right side, producing three
equations with three unknowns. A summary of the calculated percent CSA, CSB, and CSC for DS18, DS36, and
DS50 is provided in Table V. The percent of CSA-like
sequences (GlcA(β1–3)GalNAc4S(β1–4)) is observed to
increase, from DS18 to DS36 to DS50, and that for CSB
(IdoA(α1–3)GalNAc4S(β1–4)) to decrease. The absence
of CSC-like sequences (GlcA(β1–3)GalNAc6S(β1–4)) is
observed.
Although the level of CSC-like sequences for the CSC
Δdp4 oligosaccharides in Table V is 58.18%, the level of
ΔHexAGalNAc6S disaccharides in Table VI is 78.88%. The
mass spectral method compares ion signatures for a given
sample against those of the standard CS preparations. The
method thus serves as a means of comparing a series of
samples but does not provide absolute quantification of the
levels CS repeats. The CSC sample contains significantly
lower content of ΔHexAGalNAc6S (78.88%) than CS6
(93.97%). CS6 is therefore used as a mass spectral standard
against which CSC is compared. It is important to emphasize that the trend is the same for the two sets of data. Measurement of relative changes of glycoform expression
Table V. Percent compositions for glycoforms of CS/DS Δdp4, calculated
from tandem mass spectrometric signature ion abundances
1
2
3
CSA-like
GlcAGalNAc4S
CSB-like
IdoAGalNAc4S
CSC-like
GlcAGalNAc6S
CSC
41.81 ± 1.72
0 ± 0.33
DS18
0.85 ± 1.42
99.15 ± 0.35
58.18 ± 0.22
0 ± 0.14
DS36
11.21 ± 1.31
88.79 ± 0.41
0 ± 0.13
DS50
25.66 ± 1.22
74.35 ± 0.35
0 ± 0.15
17.92 ± 0.41
ACD
50.94 ± 1.35
31.13 ± 0.23
CD
35.92 ± 1.22
64.08 ± 0.28
0 ± 0.47
SD
21.56 ± 1.73
74.51 ± 0.47
3.92 ± 0.36
CS6 was used as the CSC-like standard.
between a given sample and the CS standards is the most
appropriate use of the mass spectral method.
Disaccharide analysis
Disaccharide analysis of CS/DS standards and unknown
samples was achieved by exhaustive digestion using chondroitinase ABC, subsequent fluorescent derivatization
using 2-aminoacridone (AMAC) and capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection to analyze the disaccharides (Militsopoulou et al.,
2002). Migration times of Δ-disaccharides generated from
standard CS preparations CSA, CSB, CSC, and those from
DS samples and decorin were verified against those of
AMAC-derivatized commercial disaccharide standards. An
AMAC-derivatized trisulfated disaccharide derived from
heparin (HSIS) was used as an internal standard for each
run, as it has a unique migration time compared with the
CS/DS samples analyzed. Tables VI and VII list the Δ–disaccharide composition of the whole polymer chain and the
dp6 fraction, respectively. Percentage Δ-disaccharide compositions were calculated using the CE fluorescence peak
areas, normalized to the area of the internal standard.
Partial chondroitinase ABC digestion was used to produce oligosaccharide mixtures for subsequent mass spectral
analysis. Although the distribution of oligosaccharides was
dependent to some extent on the sample, the dp4 and dp6
fractions are representative of the glycoform distribution of
the entire CS/DS chain. This can be seen from the good
agreement in disaccharide analysis values obtained from
isolated dp6 fractions versus those of the entire CS/DS
chain from which they were derived (Tables VI and VII).
For the GlcA-containing standards, CSA is shown to
contain 94.65% ΔHexA-GalNAc4S and 5.34% ΔHexAGalNAc6S, whereas CSC is a mixture of 17.45% ΔHexAGalNAc4S, 78.88% ΔHexAGalNAc6S, and 3.66% of the
2-sulfated disaccharide, ΔHexA2SGalNAc6S (Table VI).
The CS6 sample contains 93.97% ΔHexAGalNAc6S, significantly greater than the value observed for the commercial
CSC, a trend that was observed in the signature ion plots
for the Δdp4 and Δdp6 oligosaccharides (Figure 6). Thus,
Table VI. CE-LIF percentage compositions of ΔHexAGalNAc4S, ΔHexAGalNAc6S, ΔHexA2SGalNAc4S, and ΔHexA2SGalNAc6S after an exhaustive
digestion of the whole DS polymer chain
1
2
3
4
ΔHexAGalNAc4S
ΔHexAGalNAc6S
ΔHexA2SGalNAc4S
ΔHexA2SGalNAc6S
CSA
94.65 ± 0.29
5.34 ± 0.02
0
0
CSB
91.10 ± 0.39
7.10 ± 0.03
1.79 ± 0.04
0
CSC
17.45 ± 0.17
78.88 ± 0.66
0
3.66 ± 0.02
CS6
6.03 ± 0.07
93.97 ± 0.7
0
0
DS18
95.51 ± 1.00
2.65 ± 0.13
1.84 ± 0.02
0
DS36
94.39 ± 1.21
1.98 ± 0.09
3.69 ± 0.02
0
DS50
84.16 ± 2.03
13.69 ± 0.27
2.14 ± 0.03
0
ACD
60.77 ± 0.25
39.23 ± 0.16
0
0
CD
86.75 ± 0.08
9.64 ± 0.01
3.61 ± 0.01
0
SD
79.39 ± 0.01
15.59 ± 0.01
5.01 ± 0.06
0
Percentage Δ-disaccharide compositions were calculated using CE fluorescence peak areas normalized to the area of the internal standard.
509
M.J.C. Miller et al.
Table VII. CE-LIF percent compositions of CS/DS dp6 fraction after an exhaustive digestion using chondroitinase ABC
1
2
3
4
ΔHexAGalNAc4S
ΔHexAGalNAc6S
ΔHexA2SGalNAc4S
ΔHexA2SGalNAc6S
0
CSA
93.79 ± 0.11
6.20 ± 0.00
0
CSB
93.52 ± 0.19
5.43 ± 0.01
1.05 ± 0.01
0
CSC
20.07 ± 0.21
76.42 ± 0.87
0
3.51 ± 0.03
CS6
10.64 ± 0.01
89.36 ± 0.07
0
0
DS18
95.95 ± 0.76
0.85 ± 0.02
3.21 ± 0.01
0
DS36
93.07 ± 0.24
3.02 ± 0.03
3.90 ± 0.02
0
DS50
91.67 ± 0.17
6.84 ± 0.04
1.49 ± 0.03
0
Percentage Δ-disaccharide compositions were calculated using CE fluorescence peak areas normalized to the area of the internal standard.
the higher abundance of GlcA(β1–3)GalNAc6S(β1–4) in
CS6 compared with that in the CSC standard from the MS ion
signature data is supported by the CE-based Δ-disaccharide
analyses of the abundances of the ΔHexAGalNAc6S
disaccharides. Likewise, lower abundances of GlcA(β1–3)
GalNAc4S(β1–4) signature ions in CS6 relative to commercial CSC is reflected by the lower percentage of
ΔHexAGalNAc4S in the CE-LIF data.
Discussion
The use of MS ion signatures to quantify CSA-, CSB-, and
CSC-like repeats in CS/DS oligosaccharides allows the
comparison of sulfation pattern and epimerization state
among samples derived from different tissue contexts or
purification schemes. Because the mass spectral ion signatures of each are measured against the same standard CS/
DS preparations, it is possible to compare glycoform distributions among a series of GAG or PG samples derived
from different environments. The method entails digestion
with a single nonspecific enzyme, a single stage of chromatography followed by mass spectral analysis. We have also
used this approach in an LC–tandem MS platform
(Hitchcock et al., in press). This platform is limited to the
analysis of tetrasaccharides, and the present work extends
the analysis to hexasaccharides.
Decorins with GAG chains containing high DS content
have been shown to be expressed in articular cartilage
(Rosenberg et al., 1985; Sampaio et al., 1988; Cheng et al.,
1994). The activity of D-glucuronyl C5-epimerase is high in
fibroblasts, intermediate in articular cartilage, and low in
nasal cartilage (Tiedemann et al., 2001); the results of this
work (Table V), showing that levels of CSB-like repeats are
generally higher for the dermal DS than for articular cartilage decorins, are consistent with this trend in enzyme activities. The results for all decorin samples tested indicate
significant abundances of CSB-like (IdoA(α1–3)Gal
NAc4S(β1–4)) repeats from 31.1 to 74.5% (Table V).
Dermal DS samples show a consistent decrease in total
CSB-like repeats, from DS18 to DS50. DS chains are purified from skin, and the associated numeral refers to the percent ethanol at which the chains precipitated (Davidson
et al., 1956; Fransson and Roden, 1967a). Previous reports
have indicated that DS samples contain high levels of
510
IdoA-GalNAc4S repeats (Hoffman et al., 1957; Fransson
and Roden, 1967b), with lower levels of IdoA2S-Gal
NAc4S (Coster et al., 1975) and GlcA(β1–3) Gal
NAc6S(β1–4) repeats (Fransson, 1968). The disaccharide
analysis of the present DS samples shows that
ΔHexAGalNAc4S comprises 84.1–95.5% of the disaccharides formed by chondroitinase ABC digestion but that a
significant percentage of ΔHexAGalNAc6S is also detected
(Table VI). The MS ion signatures indicate that 74.3–99.1%
of the oligosaccharides have CSB-like IdoA(α1–3)-Gal
NAc4S(β1–4) character (Table V, column 2) and that CSClike GlcA(β1–3)GalNAc6S(β1–4) repeats are not detected.
It must be emphasized that the MS measurements determine the degree of similarity of oligosaccharides from an
unknown sample with those from standard CS/DS preparations. Thus, each unknown sample is compared against the
same standard compounds and, by extrapolation, to other
unknowns. Because only CSA, CSB, and CSC were used as
standards, it remains possible that low levels of other glycoforms present in the samples remain undetected. For
instance, low levels of IdoA(α1–3)GalNAc6S(β1–4) repeats,
known to be present in mammalian tissue (Bao et al., 2005),
are possibly detected as CSB-like ions. Although it has 6sulfation, MS detects the signature ion for epimerization
more than the sulfation position. At this juncture, this proposal remains to be proven until IdoA(α1–3)Gal
NAc6S(β1–4) standard becomes available. Moreover, oligosaccharides containing both 6- and 4-sulfation on the
same chain would also need an appropriate standard to
accurately identify and quantify them.
Glycans released from biological sources are typically
complex mixtures of glycoforms. Because the purification is
difficult and time consuming, methods are needed to characterize mixtures directly. By determining the compositions
of glycoforms, it is possible to profile expressed glycans as a
function of changes to the biological context. The present
work applies tandem MS analysis to CS/DS glycoforms.
Using this method, product ion profiles obtained from
unknown CS/DS samples, consisting of mixtures of glycoforms, are compared against those obtained from purified
standard preparations. Thus, the degree of similarity of the
CS/DS oligosaccharides to the standards is quantified. In
this manner, information on sulfation position and uronic
acid epimerization is produced in a single measurement.
In future work, these concepts will be developed into an
MS of CS/DS
LC/MS platform for glycomics profiling so as to streamline
the workflow. Thus, partially depolymerized CS/DS oligosaccharides will be separated using high-performance
size-exclusion chromatography with online tandem MS detection. In principle, the concept demonstrated for CS/DS oligosaccharides should be applicable to any class of glycans.
The nature of the separation system will change depending
on the chemistry of the glycan class in question, and the
structural detail produced will depend on the nature of the
standards against which unknown glycoconjugate samples
are compared.
Materials and methods
CS type A (GlcA, GalNAc4S), CSB (IdoA, GalNAc4S),
CSC (GlcA, GalNAc6S), and chondroitinase ABC were
obtained from Seikagaku America/Associates of Cape Cod
(Falmouth, MA). Purified CS Δ-disaccharide standards
were purchased from V-Labs (Covington, LA). Bovine
articular cartilage decorin was purchased from Sigma
Chemical (St. Louis, MO). DS samples were prepared by
precipitation from calcium acetate/acetic acid solution with
ethanol. AMAC was purchased from Fluka Chemical
(Milwaukee, WI), sodium cyanoborohydride was from Aldrich Chemical (Milwaukee, WI), and cellulose spin columns
were purchased from Harvard Apparatus (Holliston, MA).
Partial digestion of chondroitin and dermatan sulfate
The method for digestion was described previously (Zaia
et al., 2003). Briefly, 100 μg of CS/DS was mixed with 10 μL
of water, 2 μL of 1 M Tris–HCl buffer (pH 7.4), 1 μL of 1 M
NH4OAc, and 4 mU of chondroitinase ABC and was
digested at 37°C. After 8 min (30% digestion), the digestion
was terminated by boiling for 2 min. Oligosaccharides were
fractionated using a Superdex Peptide 3.2/30 column
(Amersham Biosciences, Piscataway, NJ), which was equilibrated in 10% aceteonitrile, 0.1 M ammonium acetate at
100 μL/min, with detection at 232 nm.
Electrospray MS
Mass spectra were acquired in the negative mode using an
Applied Biosystems/MDS-Sciex (Toronto, Canada) API
QSTAR Pulsar i quadrupole orthogonal time-of-flight
mass spectrometer fitted with a nanospray ion source. Samples were dissolved in 10% isopropanol and diluted with
10% isopropanol and 0.1% NH4OH to achieve a 1–5 pmol/
μL solution. Aliquots of 5 μL were sprayed into the mass
spectrometer using 1 μm orifice nanospray tips pulled inhouse using a capillary puller (Sutter Instrument P 80/PC
micropipette puller; San Rafael, CA). An ionization potential of –1150 V produced a steady ion signal, and all spectra
were calibrated externally. The collision energies were set so
that the precursor ion remained the most abundant. For
CID, these were best obtained at collision energy of –18
and –16 V for the Δdp4 and Δdp6 series, respectively.
Exhaustive digestion of CS/DS
To 100 μg of CS/DS was mixed 10 μL of water, 2 μL of 1 M
Tris–HCl buffer (pH 7.4), 1 μL of 1 M NH4OAc, and 20
mU of chondroitinase ABC, and digestion was allowed to
proceed at 37°C. After 12 h, an additional 20 mU of chondroitinase ABC was added. The mixture was allowed to
digest for 24 h after which the reaction was stopped by boiling the mixture for 2 min. The disaccharide collected after
size-exclusion chromatography was derivatized with AMAC.
AMAC derivatization
Derivatization with AMAC was performed following the
procedure described by Militsopoulou (Militsopoulou
et al., 2002). To the lyophilized disaccharide CS/DS (at
least 100 pmol) was added 5 μL of 0.1 M AMAC solution
in acetic acid : dimethyl sulfoxide (DMSO) (3:17, v/v) and
5 μL of a freshly prepared 1 M NaBH3CN solution in water.
The mixture was vortexed for 3 min and was incubated at
45°C for 4 h after which 50 μL of DMSO was added. Excess
reagent was removed using cellulose spin columns.
CE analysis
The AMAC-derivatized DS samples were analyzed using a
Beckman Coulter (Fullerton, CA) P/ACE MDQ capillary
electrophoresis instrument. The uncoated, fused silica capillary tube (75 μm ID, 60 cm total length) was regenerated
with 0.1 M HCl, 0.1 M NaOH, and HPLC grade water
before each run, and analyses were performed using 50 mM
NaH2PO4 buffer, pH 3.5, at 30 kV at reverse polarity and
detection using the AMAC chromophore at 254 nm. Distinct separation of the CS/DS isomers was obtained in the
reverse polarity, and reproducibility was also obtained
when fresh buffer solution was used for each run. An HSIS
was used as an internal standard for each run, as it has a
unique migration time compared with the CS/DS samples.
Acknowledgments
This work was supported by NIH grants P41RR10888 and
R01HL74197.
Conflict of interest statement
None declared.
Abbreviations
ACD, bovine articular cartilage decorin; AMAC, 2aminoacridone; CD, human cervix decorin; CE, capillary
electrophoresis; CID, collision-induced dissociation; CS,
chondroitin sulfate; CSA, chondroitin sulfate type A; CSB,
chondroitin sulfate type B; CSC, chondroitin sulfate type
C; dp, degree of polymerization; DS, dermatan sulfate; ESI,
electrospray; FGF, fibroblastic growth factor; GAG, glycosaminoglycan; HSIS, AMAC-derivatized trisulfated disaccharide
derived
from
heparin;
LC,
liquid
chromatography; LIF, laser-induced fluorescence; MS,
mass spectrometry; PG, proteoglycan; RPIP-HPLC,
reversed-phase ion pairing high-performance liquid chromatography; SD, bovine sclera decorin; Δ, 4,5-unsaturated
uronic acid.
511
M.J.C. Miller et al.
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