Tags for the stable isotopic labeling of carbohydrates and quantitative analysis by mass spectrometry

Article abstract of DOI:10.1021/ac070581b

Although stable isotopic labeling has found widespread use in the proteomics field, its application to carbohydrate quantification has been limited. Herein we report the design, synthesis, and application of a novel series of compounds that allow for the incorporation of isotopic variation within glycan structures. The novel feature of the compounds is the ability to incorporate the isotopes in a controlled manner, allowing for the generation of four tags mat vary only in their isotopic content. This allows for the direct comparisons of three samples or triplicate measurements with an internal standard within one mass spectral analysis. Quantitation of partially depolymerized glycosaminoglycan mixtures, as well as N-linked glycans released from fetuin, is used to demonstrate the utility of the tetraplex tagging strategy.

Full text of DOI:10.1021/ac070581b

Anal. Chem. 2007, 79, 5777-5784
Tags for the Stable Isotopic Labeling of
Carbohydrates and Quantitative Analysis by Mass
Spectrometry
Michael J. Bowman and Joseph Zaia*
Department of Biochemistry, Center for Biomedical Mass Spectrometry, Boston University School of Medicine,
Boston, Massachusetts 02118
These limitations require the use of methods that allow for
the analysis of small quantities of heterogeneous biological
samples. Mass spectrometry is widely used to determine the
carbohydrate components within a biological system.9-15 Accurate
quantification of glycoconjugate glycans among different samples
will enable improved understanding of human disease processes.
Such quantification remains a difficult task due to instrumental
and sample variability. The feasibility of stable isotope glycan tags
has been demonstrated by Yuan et al.16 and Hitchcock et al.17 in
which one isotopically varied reductive amination tag was used
to quantify the relative amount of carbohydrate present in samples
using liquid chromatography/mass spectrometry (LC/MS). These
duplex tags require multiple LC/MS runs to obtain statistically
relevant data. Hsu et al. used a synthesized affinity tag that
incorporates a biotin moiety and allows for the incorporation of a
single deuterium atom via reduction with sodium borodeuteride;18
however, the mass shift of 1 Da may not suffice for quantitation
due to the overlapping isotopic envelopes. The present work
describes newly synthesized tags, capable of being modified with
four isotope-enriched variants, allowing for the determination of
statistically relevant data in a single experiment (Scheme 1). The
defining feature of the tags is the ability to incorporate multiple
isotopic labels as distinct modules, thereby allowing for the direct
comparison of multiple samples using mass spectrometry.
Isotopic labeling has been applied in many forms to samples
in the proteomics field;19,20 however, isotope labels for carbohy-
drates is only beginning to find use.16-18 Experience with the ICAT
Although stable isotopic labeling has found widespread
use in the proteomics field, its application to carbohydrate
quantification has been limited. Herein we report the
design, synthesis, and application of a novel series of
compounds that allow for the incorporation of isotopic
variation within glycan structures. The novel feature of
the compounds is the ability to incorporate the isotopes
in a controlled manner, allowing for the generation of four
tags that vary only in their isotopic content. This allows
for the direct comparisons of three samples or triplicate
measurements with an internal standard within one mass
spectral analysis. Quantitation of partially depolymerized
glycosaminoglycan mixtures, as well as N-linked glycans
released from fetuin, is used to demonstrate the utility of
the tetraplex tagging strategy.
Most nuclear, cytosolic, membrane-bound, and secreted pro-
teins are glycosylated.1 Glycoconjugate glycans serve structural
functions,2 play roles in protein folding, turnover, and secretion,3
mediate cell growth4,5 and adhesion, and serve as elements for
bacterial or viral recognition and invasion.6,7 Despite their preva-
lence in biological systems, a detailed understanding of their
functions remains elusive, due in part to the nature of their non-
template-driven biosynthesis. The structures of glycoconjugate
glycans depend on the action of glycosyltransferase enzymes and
the availability of nucleotide sugar precursors.8 Because some of
the individual biosynthetic reactions do not go to completion,
expressed glycans consist of a distribution of related glycoforms
built on a common core structure that modify a given aglycon
(amino acid residue or lipid). Their heterogeneous nature, where
glycoform expression patterns vary by tissue localization and
temporal factors, such as development or disease states, also pose
analytical challenges.
(9) Haslam, S. M.; North, S. J.; Dell, A. Curr. Opin. Struct. Biol. 2006, 16,
584-591.
(10) Dwek, M. V.; Brooks, S. A. Curr. Cancer Drug Targets 2004, 4, 425-442.
(11) Harvey, D. J. Expert Rev. Proteomics 2005, 2, 87-101.
(12) Yu, Y.; Sweeney, M. D.; Saad, O. M.; Crown, S. E.; Hsu, A. R.; Handel, T.
M.; Leary, J. A. J. Biol. Chem. 2005, 280, 32200-32208.
(13) Saad, O. M.; Ebel, H.; Uchimura, K.; Rosen, S. D.; Bertozzi, C. R.; Leary, J.
A. Glycobiology 2005, 15, 818-826.
(14) Zhang, J.; Xie, Y.; Hedrick, J. L.; Lebrilla, C. B. Anal. Biochem. 2004, 334,
20-35.
(15) Madera, M.; Mechref, Y.; Klouckova, I.; Novotny, M. V. J. Chromatogr., B:
Anal. Technol. Biomed. Life Sci. 2007, 845, 121-137.
(16) Yuan, J.; Hashii, N.; Kawasaki, N.; Itoh, S.; Kawanishi, T.; Hayakawa, T. J.
Chromatogr., A 2005, 1067, 145-152.
* To whom correspondence should be addressed. E-mail: jzaia@bu.edu.
Phone: 617-638-6762. Fax: 617-638-6761.
(1) Apweiler, R.; Hermjakob, H.; Sharon, N. Biochim. Biophys. Acta 1999, 1473,
4-8.
(2) Iozzo, R. V. Annu. Rev. Biochem. 1998, 67, 609-652.
(3) Helenius, A.; Aebi, M. Science 2001, 291, 2364-2369.
(4) Varki, A.; Cummings, R.; Esko, J.; Freeze, H.; Hart, G.; Marth, J. Proteoglycans
and Glycosaminoglycans, in Essentials of Glycobiology; Cold Spring Harbor
Press: Plainview, NY, 1999.
(17) Hitchcock, A. M.; Costello, C. E.; Zaia, J. Biochemistry 2006, 45, 2350-
2361.
(5) Conrad, H. Heparin Binding Proteins; Academic Press: New York, 1998.
(6) Smith, A. E.; Helenius, A. Science 2004, 304, 237-242.
(7) Marsh, M.; Helenius, A. Cell 2006, 124, 729-740.
(8) Caffaro, C. E.; Hirschberg, C. B. Acc. Chem. Res. 2006, 39, 805-812.
(18) Hsu, J.; Chang, S. J.; Franz, A. H. J. Am. Soc. Mass Spectrom. 2006, 17,
194-204.
(19) Lill, J. Mass Spectrom. Rev. 2003, 22, 182-194.
(20) Ong, S. E.; Mann, M. Nat. Chem. Biol. 2005, 1, 252-262.
10.1021/ac070581b CCC: $37.00 © 2007 American Chemical Society
Published on Web 07/03/2007
Analytical Chemistry, Vol. 79, No. 15, August 1, 2007 5777

Scheme 1. Representation of Tetraplex Tags and Experimental Flow Chart and Sythesized
Compounds Indicating Potential Sites for Isotopic Modification
reagent21 has shown chromatographic resolution of the isotopically
varied analytes22 and fragmentation of the tag.23 These problems
have been overcome by the incorporation of 13C in place of
deuterium and an acid-cleavable linker to the biotin, which reduces
the size and fragmentation complexity of the tag.24,25
This work builds on knowledge obtained from proteomics into
the design of tags utilizing chemistry appropriate for released
glycans, compatible with tandem MS, and flexible to allow features
such as chromophores or affinity handles. Deuterated tags were
designed for use with widely accepted reductive amination
chemistry26,27 and may later be modified for use with 13C, 15N
isotopes or to incorporate affinity handles. The modules are
connected with amide bonds that are less labile than glycosidic
bonds and are unlikely to fragment under conditions used for
glycan tandem MS. They incorporate a fluorophore useful for UV-
or fluorescence-based detection and absolute quantification. Three
distinct modules create four different isotopic variations of the
tags.
Distinct, equivalent, and robust modules preserve the isotopic
information throughout the analysis process. The points of isotopic
incorporation in each module are illustrated in Scheme 1, where
each deuterium is directly bound to the carbon atoms highlighted
in green (+4), red (+8), and blue (+12). First, incorporation of
an aromatic amine allows for the facile detection of carbohydrates
that generally lack strong UV absorbance; second, it provides a
reactive group necessary to introduce the tag to the reducing end
of glycans using well-established reductive amination protocols,28
where each carbohydrate will be labeled under mild conditions
that have been shown to produce high reductive amination yields
with minimal side reactions.28 An increase in the hydrophobicity
was also desirable as this has been shown to increase the ability
(21) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R.
Nat. Biotechnol. 1999, 17, 994-999.
(22) Zhang, R.; Sioma, C. S.; Wang, S.; Regnier, F. E. Anal. Chem. 2001, 73,
5142-5149.
(23) Borisov, O. V.; Goshe, M. B.; Conrads, T. P.; Rakov, V. S.; Veenstra, T. D.;
Smith, R. D. Anal. Chem. 2002, 74, 2284-2292.
(24) Hansen, K. C.; Schmitt-Ulms, G.; Chalkley, R. J.; Hirsch, J.; Baldwin, M. A.;
Burlingame, A. L. Mol. Cell. Proteomics 2003, 2, 299-314.
(25) Li, J.; Steen, H.; Gygi, S. P. Mol. Cell. Proteomics 2003, 2, 1198-1204.
(26) Anumula, K. R. Anal. Biochem. 2000, 283, 17-26.
(27) Anumula, K. R. Anal. Biochem. 2006, 350, 1-23.
(28) Bigge, J. C.; Patel, T. P.; Bruce, J. A.; Goulding, P. N.; Charles, S. M.; Parekh,
R. B. Anal. Biochem. 1995, 230, 229-238.
5778 Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

Scheme 2. (A) Solid-Phase Synthetic Scheme for Compounds 2-5 and (B) Solution-Phase Synthetic
Scheme for Compounds 6-8
of carbohydrates to ionize.29 The incorporation of charged moieties
to the tag was considered undesirable as it would be expected to
alter the glycan product ion patterns and may be more likely to
undergo partial or complete loss of the tag. The final consider-
ations were ease of synthesis, expense, and availability of isoto-
pically enriched starting materials. The design requires that at
least two pieces of the tag be bifunctional for the incorporation of
additional modules of the tag. The use of bifunctional modules
allows for the propagation of the tag to the desired length and
isotopic content, as well as the ability to perform the synthesis
using a solid support.
The present work describes the application of tetraplex
glycomics tags to glycosaminoglycan and N-linked oligosaccha-
rides. A reference glycan mixture is labeled with the light form
of the tag, and three unknown glycan mixtures are labeled with
the heavy forms. The MS dimension of tagged tetraplex glycan
mixtures determines the monosaccharide compositions of the ions
and their quantities relative to the standard mixture. Each ion in
the MS mode reflects a mixture of isobaric glycoforms. The
abundances of tandem mass spectrometric product ions reflect
the glycoforms that compose each precursor ion. Thus, the
tandem mass spectra provide a means of following the expression
of glycoform fine structure as a function of biological change. For
glycan classes where well-defined standards exist, the product ion
abundances determine the glycoform mixture percentages. Frag-
mentation characteristics of glycoconjugate glycans vary signifi-
cantly based on compound class. In particular, the presence of
acidic functional groups strongly influences product ion pattern.30,31
The tags were therefore tested on neutral, sialylated, and sulfated
glycan classes.
resin attachment point within their structure were synthesized
using a solution-phase approach (Scheme 2B and Supporting
Information). Nucleophilic attack of an amine on a succinic
anhydride provides a monofunctional intermediate that is then
reacted with benzene diamine to yield the desired products. The
starting materials were purchased commercially with the desired
isotopic content; see Supporting Information for details. For testing
purposes, tags 2-8 were synthesized in +0 form. After testing,
tag 3 was synthesized in four stable deuterated forms (+0, +4,
+8, +12).
Chondroitin sulfate (CS) types A, B, and C were partially
depolymerized using chondroitinase ABC and the oligomers
fractionated using high-performance size exclusion chromatog-
raphy (SEC) as previously described.17 CS oligosaccharides types
A, B, and C were reductively aminated with synthesized com-
pounds 2-8, as well as anthranilic acid (1); for detailed
procedures see Supporting Information. The derivatized CS
oligosaccharides were purified using a cellulose microspin car-
tridge as previously described.17
The N-linked glycans of fetuin, in 100-, 200-, 300-, and 400-
pmol quantities, were released by enzymatic treatment with
N-glycanase (PROzyme, San Leandro, CA), in triplicate, according
to the protocol provided by the manufacturer. An aliquot was
removed and used to confirm glycan release by SDS-PAGE. The
detergent, buffer, and protein components of the resultant digests
were removed by solid-phase extraction using porous graphitized
carbon columns (500 mg of packing material, Thermo Fisher
Scientific, Waltham, MA). The glycan-containing fraction was
derivatized with stable isotopically labeled tags; excess tag was
removed by chloroform extraction, followed by C18 chromatog-
raphy in a spin column format. The resultant labeled fraction was
introduced by nanospray ionization using a Qstar mass spectrom-
eter for detailed procedures see Supporting Information.
Identical conditions for derivatization were used for all tags
and glycans, and the resulting data incorporate additional factors,
such as labeling efficiency, sample recovery from cleanup and
chromatography, and ionization efficiency. No appreciable quantity
(<5%) of unlabeled material was observed in the mass spectrum.
EXPERIMENTAL SECTION
Tags consisting of amino acid modules 2-5 were synthesized
using standard solid-phase methodologies, in high yield and purity
(Scheme 2A and Supporting Information). Tags 6-8 lacking a
(29) Harvey, D. J. J. Am. Soc. Mass Spectrom. 2000, 11, 900-915.
(30) Seymour, J. L.; Costello, C. E.; Zaia, J. J. Am. Soc. Mass Spectrom. 2006,
17, 844-854.
(31) Zaia, J.; Miller, M. J. C.; Seymour, J. L.; Costello, C. E. Submitted.
Analytical Chemistry, Vol. 79, No. 15, August 1, 2007 5779

Figure 1. (A) Nanospray ion abundances for 600 pmol (diluted to 5 pmol/µL) of CSA dp4 labeled with tags 1-8. (B) Comparison of tandem
mass spectrometric product ion abundances for CSA dp4 labeled with tags 1-8. (1 black, 2 red, 3-green, 4 yellow, 5 blue, 6 pink, 7 cyan, 8
gray) (C) Structures of anthranilic acid (1) and compounds 2-8.
Detailed procedures can be found in the Supporting Information.
This is consistent with published results demonstrating high yields
using reductive amination chemistry.28
fitting these criteria were synthesized and tested for viability in a
mass spectrometric experiment by labeling glycan preparations.
Glycosaminoglycans are linear biopolymers, and the CS class
consists of repeating disaccharide units of N-acetylgalacto-
samine and hexuronic acids, with some variations in the
structural elements. They exist primarily in three forms: CSA,
(GlcAâ3GalNAc4Sulfateâ4)n, CSB (IdoAR3GalNAc4Sulfateâ4)n
and CSC, (GlcAâ3GalNAc6Sulfate)n. Variations in the quantities
of each CS type have been implicated in disease states, such as
osteoarthritis.33 Intact glycosaminoglycans are too large and
heterogeneous to analyze; therefore, they are enzymatically
depolymerized into smaller chain lengths for analysis. Chondroitin
lyase enzymes cleave CS chain N-acetylgalactosamine bonds using
an eliminative mechanism to produce a 4,5-unsaturated (∆-
unsaturated) uronic acid on the nonreducing terminus of the
products. The ∆-unsaturation creates a unique chromophore,
allowing monitoring of the reaction at a wavelength of 232 nm.
This chromophore also allows for the quantitative comparison of
native and labeled glycosaminoglycans. A typical reaction is
stopped at 30% digestion, as this sufficiently reduces the complex-
ity of the system, while still providing a distribution of larger
oligomers containing the structural information necessary for
determination of the glycoforms present.
Oligosaccharides were analyzed using a quadrupole orthogonal
time-of-flight (Q-oTOF) mass spectrometer (Applied Biosystems/
MDS Sciex Qstar/Pulsar i) using nanospray32 ionization. Nano-
spray tip position was monitored using a pair of cameras and a
grid on the video display to maintain the distance, angle, and
alignment of the tip. Optimized conditions for the analysis of
glycosaminoglycans were used for mass spectral analysis. Frag-
mentation of the resultant tagged glycans was also determined,
using consistent collision energy (CAD ) 2, CE ) -20) to
determine the stability and fragmentation patterns of the tag-
incorporated glycans.
RESULTS AND DISCUSSION
A series of glycomics tags were designed that incorporate
stable isotope modules into the oligosaccharide structure. The
design allows for the construction of four chemically identical tags
differing only in the isotopic content within the labeling agent.
Additional features incorporated into the design were UV-active
groups to allow for spectrophotometric detection, cost effective-
ness, commercial availability of starting materials, and ease of
synthesis. Of the potential candidates, seven compounds (2-8)
(32) Wilm, M. S.; Mann, M. Int. J. Mass Spectrom. Ion Processes 1994, 136, 167-
(33) Shinmei, M.; Miyauchi, S.; Machida, A.; Miyazaki, K. Arthritis Rheum. 1992,
35, 1304-1308.
180.
5780 Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

Figure 3. (A) Negative ion nanospray MS of four tetraplex labeled
releases of N-linked glycans from bovine fetuin in quantities of 100-,
200-, 300-, and 400-pmol glycoprotein quantities labeled with d0, d4,
d8, and d12, respectively. (B) Comparison of MS ion abundances from
N-linked glycans from triplicate releases of labeled fetuin samples.
and CSC) as well as the C3 ion (not detected). In addition, 0,2X3
cross-ring cleavage is observed for all tags except 6, where no
cross-ring cleavage was observed for all three glycoforms. In the
case of 6, there was a drastic increase in the abundance of C2,
Y2, and B3 ions observed, with concomitant decrease of the Y1
ion. Tags 2-5, 7, and 8 have suitable fragmentation patterns for
use in a tandem MS experiment to gain more structural and
quantitative information about the glycan analyte from the frag-
ments that contain the reducing end of the carbohydrate.
Compound 3 was chosen for further investigations based on a
balance of mass spectrometric performance and relatively low cost
of synthetic precursors.
The usefulness of the tetraplex tag was demonstrated first
using four different variations in the extent of enzymatic depo-
lymerization of CS mixtures, as determined by 232-nm absorbance.
A 10% digestion was labeled with d0, a 20% digestion was labeled
with d4, a 30% digestion was labeled with d8, and a 40% digestion
was labeled with d12. The SEC chromatograms (Figure 2A) show
the relative abundances of the dp2, 4, and 6 oligosaccharide
products. Figure 2B shows the mass spectrometric ion abun-
dances of the d0-, d4-, d8-, and d12-tagged dp4 oligosaccharides. As
shown in Figure 2C, the isotopic tagging procedure produces
results comparable with direct measurement of dp4 concentration
from absorbance values.
Figure 2. (A) High-performance SEC of a CSA/CSB/CSC (1:1:1)
mixture enzymatically depolymerized to varied percentages, moni-
tored at 232 nm. (B) Mass spectral data of a dp4 fraction of four CSA/
CSB/CSC (1:1:1) digestions to various levels of completion labeled
with tetraplex tags (10% completion d0, 20% completion d4, 30%
completion d8, 40% completion d12). (C) Comparison of UV data of
the native dp4 digestion product from SEC-HPLC and tetraplex tagged
dp4 fractions from digestion replicates by MS.
As shown in Figure 1A, dp4 oligosaccharides derived from CSA
labeled with the commonly used glycan label 1 showed a relatively
weak signal in comparison to the other tags. There is an increase
in ionization as the tags become larger and more hydrophobic
from 2-fold (compound 6) to between 10- (compounds 2-5, 8)
and 30 (compound 7)-fold. Similar trends for each tag were
observed in labeled preparations of CSB and CSC (data not
shown). The product ion percent total ion abundances for tagged
CSA dp4 oligosaccharides are shown in Figure 1B. All eight tags
are stable to MS and tandem MS conditions, and glycan frag-
mentation results in the observed product ions. The incorporation
of the different tags on the standard glycans showed some
differences in the fragmentation patterns observed. The use of
uncharged tags (2-8) in place of anthranilic acid (1) show a
2-fold reduction in the presence of the B1 ions (for CSA, CSB,
To demonstrate the utility of the tags for quantitation of
N-linked glycans, fetuin was treated with N-glycanase. The
Analytical Chemistry, Vol. 79, No. 15, August 1, 2007 5781

Figure 4. (A) Mass spectrum of d0 CSA, d4 CSB, d8 CSC, and d12 (1:1:1 CSA/CSB/CSC) dp4 fraction from SEC-HPLC. (B) Structure and
diagnostic fragmentation pattern of labeled CSC-dp4. C. Tandem MS of d0 CSA, d4 CSB, d8 CSC, and d12- (1:1:1 CSA/CSB/CSC) dp4 fraction.
released and isolated glycans were labeled with tag 3 and
quantified using mass spectrometry. Quantities of starting glyco-
protein of 100, 200, 300, and 400 pmol were labeled with d0, d4,
d8, and d12, respectively. As shown in Figure 3A, tagged N-linked
glycans are detected in four different isotopic forms, the ion
abundances of which determine the quantity of each composition
relative to the d0-tagged glycans. The insets show expanded m/z
clusters, thereby complicating data interpretation; the use of an
on-line LC/MS system should be employed.
The previous examples demonstrate the ability to quantitate
relative differences between samples based on composition. As
many glycan isomers can contribute to each composition, it is
beneficial to utilize labeled glycans for tandem mass spectrometric
experiments to determine the isomeric contributions for each
composition.
Ions observed in the MS mode reflect mixtures of isobaric
glycoforms, the compositions of which may be performed using
stages of tandem MS. In these experiments, all four isotopic
variants were isolated using a 10 u window. The following
experiment demonstrates that equal responses result for all four
isotopes. Chondroitinase ABC was used to depolymerize separate
samples of CSA, CSB, and CSC to 30% completion. Samples
2-
3-
ranges for Bi(NeuAc)2 (1276-84), Tri(NeuAc)2 (972-80),
3-
4-
Tri(NeuAc)3 (1070-76), and Tri(NeuAc)4 (874-80). Figure
3B graphically illustrates the ion abundances for the four glycan
compositions determined for the d0, d4, d8, and d12 glycan mixtures.
These data demonstrate the reproducible and quantitative nature
of the tagging procedure for determining N-linked glycan com-
positions relative to a d0-tagged internal standard. More complex
glycan mixtures from biological samples may have overlapping
5782 Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

containing equivalent amounts of CSA oligosaccharides were
labeled with all four isotopic tags and analyzed using tandem MS;
CSB and CSC samples were also prepared and analyzed. A 10 u
wide window was used to select all four isotopic forms of the
tagged ions, in the 2- charge state, to ensure the complete isolation
of each parent ion. The results demonstrate that the product ion
abundances are consistent across the four isotopic variants for
CSA. Similar results were obtained using the CSB and CSC
subclasses. Relative abundances for product ions from three
replicates of the entire procedure are as follows: CSA y1, 0.442 (
0.025; y3, 0.235 ( 0.009; [M - H - SO3], 0.305 ( 0.019; CSB y1,
0.077 ( 0.013; y3, 0.510 ( 0.025; [M - H - SO3], 0.412 ( 0.019;
CSC y1, 0.206 ( 0.016; y3, 0.048 ( 0.008; [M - H - SO3], 0.750
( 0.011.
Table 1
(A) Percentages of CSA, CSB, and CSC Present
in the dp4 Fraction of a 30% Depolymerization
Prepared Mixtures after Digestion and Labeling
digestion
completion (%)
% CSA
% CSB
% CSC
1:1:0
1:0:1
0:1:1
1:1:1
2:1:1
1:2:1
1:1:2
49.9 ( 1.1
54.0 ( 1.5
3.1 ( 1.7
32.0 ( 2.4
54.7 ( 4.0
24.1 ( 5.1
26.6 ( 5.1
46.0 ( 3.8
2.6 ( 2.0
50.0 ( 2.6
30.5 ( 4.4
23.3 ( 4.2
49.4 ( 2.9
24.5 ( 5.4
4.1 ( 2.8
43.4 ( 2.5
46.8 ( 2.1
37.3 ( 3.1
21.9 ( 3.2
26.5 ( 3.2
48.9 ( 3.6
(B) Percentages of CSA, CSB, and CSC Present
in the dp4 Fraction of 1:1:1 CSA/CSB/CSC Mixtures
Prepared Prior to Digestion and Labeling.
The glycomics tags facilitate analysis of isobaric glycoforms,
as shown in Figure 4. To aid in visualization of the distinct
differences in isomeric fragmentation, samples were labeled with
+0 (CSA), +4 (CSB), +8 (CSC), and +12 tags (1:1:1 of CSA/
CSB/CSC). Tandem mass spectral analysis of each type of
chondroitin allows for the assignment of diagnostic ions for each
type of chondroitin. In cases where standards exist, the distinctive
fragmentation patterns can be determined, which allows for the
quantitation of isomeric analytes. Using equations based on the
patterns of fragmentation for each class of chondroitin, one can
solve for the amount of each class present in a sample.17,34-37
Figure 4A shows a typical mass spectrum of a tagged mixture of
CS oligosaccharides. The peak cluster from m/z 624-630 corre-
sponds to the four tagged forms of CS dp4. The MS ion intensities
define the quantity of the +4, +8, and +12 relative to the +0 form.
All four forms were isolated in a single window and subjected to
tandem MS. The Y1 ion is diagnostic for CSA repeats, the Y3 for
CSB, and the M - H - SO3 for CSC.17,37 The intensities of each
of the four isotopic forms for these product ions are shown in
Figure 4C. These product ion intensities were used to determine
the CSA/CSB/CSC mixture percentages using a system of linear
equations. The system has been previously used for the compo-
sitional analysis of glycosaminoglycans using electrospray ioniza-
tion quadrupole ion trap mass spectrometry17,34-36 and Q-oTOF
mass spectrometry.37 Using the values from the pure standards
and experimentally determined values from various mixtures,
performed in triplicate within each analysis, it is possible to
determine CS glycoform mixture percentages in unknown samples.
In the present experiments, unknown samples were run in
triplicate using the +4, +8, and +12 tags using +0 CSA standard
oligosaccharides as internal standards. The final isomer percent-
ages were determined by averaging the three isotopic variations
(d4, d8, d12) of each mixture, and the standard deviation is
representative of the total experimental error from digestion
through mass spectral analysis. By inclusion of a constant CSA
internal standard (d0), it is possible to determine the absolute
quantity of sample present in the mixtures, as well as verify the
operation of the mass spectrometer via any variation of the
fragmentation pattern. The results for a series of CS oligosaccha-
ride mixtures made post-enzyme digestion are shown in Table
1:1:1 A:B:C
1:1:1 A:B:C
1:1:1 A:B:C
1:1:1 A:B:C
10
20
30
40
62.8 ( 2.2
59.0 ( 2.1
30.7 ( 3.4
34.8 ( 4.5
13.3 ( 1.5
22.2 ( 1.4
29.2 ( 4.7
26.3 ( 2.4
24.1 ( 0.6
18.8 ( 1.2
40.1 ( 4.1
38.9 ( 3.1
1A. The results demonstrate the high level of reproducibility from
analysis of experimental replicates using the isotopic tags. The
coefficients of variation are within 10% of the measured value for
all compositions greater than 30%. This low degree of variation
enables the determination of biologically produced change in CS
structure while minimizing the number of experimental subjects
required for statistical significance.
Table 1B shows results for analysis of mixtures of CS chains
made before the enzymatic digestion. The results show that the
chondroitinase ABC enzyme cleaves CSA faster, as evidenced by
the high percent of this isoform in the 10% digest. CSB appears
to be cleaved slowest among the three glycoforms. As the percent
of digestion increases, the distribution of dp4 glycoforms ap-
proaches the expected 1:1:1 ratio. In order to digest the CSB and
CSC classes more evenly, it will be necessary to add chondroiti-
nase B and ACI to the digestion mixture. These results are useful
for deriving a balance of chondroitinase enzymes that digest all
types of CS evenly.
In the current study, the lowest detection limits of labeling
and recovery were not determined, in part due to the multiple
steps of purification necessary for nanospray. An expected,
improvement in the limit of detection will come from the
development of a LC/MS system capable of removing excess tag
and reductive amination salts, thereby eliminating the need for
multiple sample preparation steps necessary for compatability
with nanospray analysis. This point is currently under further
investigation.
CONCLUSIONS
Glycoconjugate glycans released from any animal source
consist of mixtures of glycoforms including a large number of
structural isomers. It is imperative that analytical methods be
available to determine the compositions of such mixtures directly.
Accordingly, stable isotope-labeled glycomics tags were developed
in this work to facilitate reproducible and accurate determination
of glycoform mixture compositions. The tags were developed for
use with widely accepted reductive amination chemistry to achieve
derivatization in high yield. They produce quantitative results for
four samples simultaneously and facilitate the inclusion of internal
(34) Desaire, H.; Leary, J. A. J. Am. Soc. Mass Spectrom. 2000, 11, 916-920.
(35) Desaire, H.; Leary, J. A. J. Am. Soc. Mass Spectrom. 2000, 11, 1086-1094.
(36) Saad, O. M.; Leary, J. A. Anal. Chem. 2003, 75, 2985-2995.
(37) Miller, M. J.; Costello, C. E.; Malmstrom, A.; Zaia, J. Glycobiology 2006,
16, 502-513.
Analytical Chemistry, Vol. 79, No. 15, August 1, 2007 5783

performance standards. For CS oligosaccharides, standards with
known glycoform composition are available and tandem mass
spectrometry of tagged mixtures allows determination of mixture
percentages of some structural isomers. For N-linked glycans, the
tags enable precise comparison of glycoform expression among
different samples. For this glycan class, the MS data show changes
in composition, reflected by the ion abundances relative to those
of the standard mixture. Tandem MS will be useful for comparing
the glycoform mixtures that make up a given composition to that
for the standard mixture. Preliminary results of neutral glycans,
released high-mannose glycans of RNaseB, and heptamaltose,
indicate that glycans labeled with 3 can be ionized by MALDI
without fragmentation of the tag; however, further studies are
necessary to determine the effect of tagging on the ionization.
ACKNOWLEDGMENT
This work was funded by NIH Grants P41RR10888 and
R01HL74197.
SUPPORTING INFORMATION AVAILABLE
Synthesis of compounds 2-8, preparation of glycosaminogly-
can digestions, N-linked glycoprotein release protocols, labeling
protocols, and mass spectrometry conditions. This material is
available free of charge via the Internet at http://pubs.acs.org.
Received for review March 23, 2007. Accepted May 24,
2007.
AC070581B
5784 Analytical Chemistry, Vol. 79, No. 15, August 1, 2007