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Novel Tags for the Stable Isotopic Labeling of
Carbohydrates and Quantitative Analysis by Mass
Spectrometry
Michael J. Bowman and Joseph Zaia*
Boston University School of Medicine, Department of Biochemistry, Center for Biomedical Mass
Spectrometry, Boston, MA 02118
[email protected]
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
TITLE RUNNING HEAD Novel Stable Isotopic Carbohydrate Tags for MS
CORRESPONDING AUTHOR FOOTNOTE Joseph Zaia, Boston University School of Medicine,
Department of Biochemistry, Center for Biomedical Mass Spectrometry, 670 Albany St., Rm 509,
Boston , MA 02118 (p) 617-638-6762, (f) 617-638-6761
ABSTRACT 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
1
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.
KEYWORDS. Carbohydrate Quantification, Stable Isotope Tag, Glycomics
MANUSCRIPT TEXT
Introduction
Most nuclear, cytosolic, membrane bound and secreted proteins are glycosylated.1 Glycoconjugate
glycans serve structural functions,2 play roles in protein folding, turnover, and secretion,3mediate cell
growth4, 5 and adhesion, and serve as elements for bacterial or viral recognition and invasion.6, 7 Despite
their prevalence 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.
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
2
LC/MS runs to obtain statistically relevant data. Hsu et al. use a synthesized affinity tag which
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, the
use of isotope labels for carbohydrates is only beginning to find use.16-18 Experience with the ICAT
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 and/or affinity handles. Deuterated tags were designed for use with widely accepted
reductive amination chemistry26,
27
and may later be modified for use with
13
C,
15
N 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).
Incorporation of an aromatic amine allows for the facile detection of carbohydrates that generally lack
3
strong UV absorbance, secondly 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 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 considerations
were ease of synthesis, expense, and availability of isotopically-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 Nlinked oligosaccharides. 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.
Fragmentation
characteristics of glycoconjugate glycans vary significantly 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.
Experimental
4
Tags consisting of amino acid modules (2-5) were synthesized using standard solid phase
methodologies, in high yield and purity (Scheme 2A and supplemental information). Tags (6-8) lacking
a resin attachment point within their structure were synthesized using a solution phase approach
(Scheme 2B and supplemental 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
supplemental 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 chromatography (SEC) as
previously described.17 CS oligosaccharides types A, B, and C were reductively aminated with
synthesized compounds 2-8, as well as anthranilic acid (1), for detailed procedures see supplemental
information. The derivatized CS oligosaccharides were purified using a cellulose micro-spin cartridge
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 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
chromatography in a spin column format. The resultant labeled fraction was introduced by nanospray
ionization using a QSTAR mass spectrometer, for detailed procedures see supplemental information.
Identical conditions for derivatization were used for all tags and glycans and the resulting data
incorporates additional factors, such as labeling efficiency, sample recovery from cleanup and
chromatography, and ionization efficiency. No appreciable quantity (<5%) of unlabeled material was
5
observed in the mass spectrum. Detailed procedures can be found in the supplemental information.
This is consistent with published results demonstrating high yields using reductive amination
chemistry.28
Oligosaccharides were analyzed using a quadrupole orthogonal time-of-flight (Q-oTOF) mass
spectrometer (Applied Biosystems/MDS Sciex Qstar/Pulsar i) using nanospray32 ionization. Nanospray
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. Fragmentation 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 effectiveness,
commercial availability of starting materials, and ease of synthesis. Of the potential candidates, seven
compounds (2-8) 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-acetyl-galactosamine and hexuronic acids, with some variations in the structural elements. They
exist primarily in three forms: CSA, (GlcAβ3GalNAc4Sulfateβ4)n, CSB (IdoAα3GalNAc4Sulfateβ4)n
and CSC, (GlcAβ3GalNAc6Sulfate)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 (Δ-unsaturated) uronic acid on the non-reducing terminus of
6
the products. The Δ-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 complexity of the system, while still providing a distribution of larger oligomers containing the
structural information necessary for determination of the glycoforms present.
As shown in Fig. 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 Fig. 1B. All 8 tags are stable to MS and tandem MS
conditions and glycan fragmentation 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, and CSC) as well as the C3 ion (not detected). In addition,
0,2
X3 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 compound 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, 7-8) have suitable fragmentation
patterns for use in a tandem MS experiment to gain more structural and quantitative information about
the glycan analyte from the fragments 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 depolymerization 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 with d8, and a 40% digestion
was labeled with d12. The SEC chromatograms (Fig. 2A) show the relative abundances of the dp2, 4
7
and 6 oligosaccharide products. Fig. 2B shows the mass spectrometric ion abundances of the d0-, d4-,
d8-, and d12-tagged dp4 oligosaccharides. As shown in Fig. 2C, the isotopic tagging procedure produces
results comparable with direct measurement of dp4 concentration from absorbance values.
To demonstrate the utility of the tags for quantitation of N-linked glycans, fetuin was treated with NGlycanase®. The released and isolated glycans were labeled with tag 3 and quantified using mass
spectrometry. Quantities of starting glycoprotein 100 pmol, 200 pmol, 300 pmol, and 400 pmol were
labeled with d0, d4, d8, and d12, respectively. As shown in Fig. 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 ranges for Bi(NeuAc)22(1276-84), Tri(NeuAc)23- (972-80), Tri(NeuAc)33- (1070-76), and Tri(NeuAc)44- (874-80). Fig. 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 compositions relative to a d0-tagged internal standard.
More complex glycan mixtures from biological samples may have overlapping 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 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
8
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.
The glycomics tags facilitate analysis of isobaric glycoforms, as shown in Fig. 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 Fig. 4A shows a
typical mass spectrum of a tagged mixture of CS oligosaccharides. The peak cluster from m/z 624-630
corresponds 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 Fig. 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 compositional analysis of
glycosaminoglycans using electrospray ionization quadrupole ion trap mass spectrometry17, 34-36 and QoTof 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 percentages were
9
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 oligosaccharide mixtures made
post-enzyme digestion are shown in Table 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 approaches the
expected 1:1:1 ratio. In order to digest the CSB and CSC classes more evenly, it will be necessary to
add chondroitinase 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
10
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 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.
Acknowledgments. This work was funded by NIH grants P41RR10888 and R01HL74197.
Supporting Information Available Synthesis of compounds 2-8, preparation of glycosaminoglycan
digestions, N-linked glycoprotein release protocols, labeling protocols, and mass spectrometry
conditions are available.
Table 1. A. Percentages of CSA, CSB, and CSC present in the dp4 fraction of a 30% depolymerization
prepared mixtures after digestion and labeling. 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.
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FIGURE CAPTIONS
Figure 1. A. Nanospray ion abundances for 600 pmol (diluted to 5 pmol/μl) CSA dp4 labeled with tags
1-8. B. Comparison of tandem mass spectrometric product ion abundances for CSA dp4 labeled with
14
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.
Figure 2. A. High performance SEC of a CSA:CSB:CSC (1:1:1) mixture enzymatically depolymerized
to varied percentages, monitored 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.
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, d12, respectively. B. Comparison of MS ion abundances from N-linked glycans from triplicate
releases of labeled fetuin samples.
Figure 4. A. Mass spectrum of d0- CSA, d4- CSB, d8- CSC, 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, d12- (1:1:1 CSA:CSB:CSC) dp4 fraction.
Scheme 1. Representation of tetraplex tags and experimental flow chart and sythesized compounds
indicating potential sites for isotopic modification.
Scheme 2. A.) Solid-phase synthetic scheme for compounds 2-5. B.) Solution phase synthetic scheme
for compounds 6-8
Supplemental Information:
Experimental:
15
CS
type
A
((GlcAβ3GalNAc4Sulfateβ4)n),
CSB
((IdoAα3GalNAc4Sulfateβ4)n)
CSC
((GlcAβ3GalNAc6Sulfate)n), and chondroitinase ABC, B and AC1 were obtained from Seikagaku
America/Associates of Cape Cod (Falmouth, MA). Purified Δ-disaccharide standards were purchased
from V-labs (Covington, LA). 2-anthranilic acid, Methylene Chloride (MC), and Piperdine (20 % in
DMF) were purchased from Fluka Chemika (Buchs, Switzerland).
Dimethyl formamide (DMF),
ethanol (EtOH), O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 1Hydroxybenzotriazole (HOBt) , Fmoc- protected Rink resin, N-(9-Fluorenylmethoxycarbonyl)-Lalanine (Fmoc-d0-Ala), diisopropylethyl amine (DIEA), piperdine (20 % in DMF), triisopropyl silane
(TIPS), Trifluoroacetic acid
phenylenediamine,
succinic
(TFA), ninhydrin, 4-isopropylanaline, 1-1-adamantyl-ethyl amine, panhydride,
aminobenzamide,
N-(3-Dimethylaminopropyl)-N′-
ethylcarbodiimide hydrochloride (EDC), and sodium cyanoborohydride were from Aldrich Chemicals
Co (St. Louis, MO).
N-(9-Fluorenylmethoxycarbonyl)-L-alanine (Fmoc-d4-Ala) was from C/D/N
Isotopes (Quebec, Canada). Solid phase extraction tubes (1 ml) were purchased from Supelco
(Bellefonte, PA). Cellulose packing material Micro Spin Columns were purchased from Harvard
Apparatus (Holliston, MA). Peptide-N-Glycosidase F (PNGase F) was purchased from PROzyme, (San
Leandro, CA.) Porous graphitized carbon columns (SuperSil) were purchased from Thermo Fisher
Scientific (Waltham, MA). C18 Macrospin columns were purchased from Nest Group (Southborough,
MA).
In a typical reaction, Tags (2-5) were synthesized by standard manual solid phase peptide synthetic
methods. Reactions were carried out in 1 ml polypropylene syringe barrels (Supelco) utilizing a
polyethylene frit (0.2 μm). In a typical reaction sequence Rink resin (Aldrich, 0.6 mmol/g loading, 50
mg) was treated with piperdine:DMF (2:8) (1x min, 1x 20 min) to remove the Fmoc- protecting group.
After removal of the piperidine solution the resin was washed with DMF (3x 1 min), methylene chloride
(3x 1 min), ethanol (1x 3 min), methylene chloride (3x 1 min), DMF (3x 1 min) providing the resin
bound free amine. Three equivalents of Fmoc protected amino acid (d0 or d4), HBTU, HOBt, and
diisopropylethyl amine (DIEA) were preactivated for 5-8 min in DMF (0.6 ml, 300 mM final
16
concentration), after which they were added to the resin and reacted for 2 hours. After 2 hours, the
excess reagent was filtered off and the resin was washed with DMF (3x 1 min), MC (3x 1 min), and
ethanol (1x 3 min) to remove any residual reactants. The resin was rinsed with MC (3x 1 min) and
DMF (3x 1 min) in preparation for deprotection. The resin was treated with piperdine:DMF (2:8) (1x
min, 1x 20 min). After washing, the resin was ready for the addition of the next amino acid. This cycle
was repeated until the desired length had been achieved. The compounds were then cleaved from the
resin using a trifluoroacetic acid (TFA): triisopropyl silane (TIS): water (95:2.5:2.5) solution for 2 hours
under nitrogen, followed by precipitation in cold ether. The resulting compounds were purified to
homogeneity by reverse-phase HPLC utilizing a C18 column (Vydac, 10 mm ID x 250 mm) on a 5-15%
gradient of methanol:water. Purified compounds were analyzed by ESI-MS on a Quattro II mass
spectrometer to confirm their identity.
Tags lacking a terminal carboxamide (6-8) were synthesized in solution phase. In a typical reaction,
0.25 mmol of succinic anhydride was dissolved in 1.5 ml ACN, followed by addition of the desired
amine (0.25 mmol). The reaction was heated at 60˚C for 4 hrs. The resulting monoacidic compound
was purified by silica gel chromatography (ethyl acetate:hexane). The free acid was activated by N-(3Dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) in MC in the presence of 2 eq. DIEA, followed by
addition of 10 equivalents of 1,4-diaminobenzene for 2 hours at room temperature.
The desired
products were purified to homogeneity via silica gel chromatography and/or reverse-phase HPLC
utilizing a C18 column (Vydac). Purified compounds were analyzed by ESI-MS on a Quattro II mass
spectrometer to confirm their identity.
The partial digestion of chondroitin sulfate A, B, and C was achieved by the action of chondroitinase
ABC on the desired substrate was done as previously described. A complete digestion was used to
obtain the absorbance value equal to complete conversion to disaccharides. From this value a 10, 20, 30
and 40% digestion value was determined. Briefly, 10 μg of chondroitin substrate was digested in a
solution containing 5 μl 1 M Tris-HCl (pH 7.4), 1 μl 1 M NH4OAc, and 2 mU chondroitinase enzyme in
17
100 μl water. The digestion was terminated at the proper absorbance value by boiling in water for two
minutes.
All oligosaccharides were derivatized with d0-1-8, d0-, d4-, d8- or d12-3 according to the method of
Bigge et al.28 with slight modification. Briefly, CSA samples were dried in the presence of the desired
label (in 10% MeOH).
The dried sample was then dissolved in 10 μL of a reaction reagent
DMSO/glacial acetic acid (7:3) containing 1.0 M sodium cyanoborohydride. The glycan solutions were
vortexed then centrifuged and incubated at 65 °C for 3 h. Excess reagents were removed with cellulose
microspin columns as follows. The column was first hydrated with five 200 μL volumes of water,
washed with five 200 μL volumes of 30% acetic acid solution, and then equilibrated with three 200 μL
volumes of acetonitrile. The derivatized reaction mixture was applied to the column allowing 10 min for
it to adsorb to the cellulose. Excess reagent was washed off with three 200 μL volumes of acetonitrile
followed by two 200-μL volumes of 96% acetonitrile. The derivatized glycan was then eluted with two
100-μL volumes of water and dried.
Derivatized glycan samples were fractionated using high
performance SEC. Briefly, the column (Superdex Peptide 3.2/30, Amersham Biosciences, Piscataway,
NJ) was equilibrated in 10% acetonitrile, 0.05 M ammonium formate solution at 40 μL/min and the
oligosaccharide mixture (10 μL) was injected with UV detection at 232 nm. Samples were lyophilized
three times in equal amounts of water to remove the volatile salts prior to MS analysis.
The N-Linked glycans of bovine fetuin (Sigma, St Louis, MO) were released from their proteins by
the action of N-Glycanase® (Peptide-N-Glycosidase F, PROzyme, San Leandro, CA) according to the
manufacturer’s protocol. Briefly, 100 pmol (6 μg), 200 pmol (12 μg), 300 pmol (18 μg), and 400 pmol
(24 μg) quantities of fetuin were dissolved in 45 μl reaction buffer (10 mM Tris-HCl, pH 8.0), 2.5 μl of
denaturation solution was added (2% SDS, 1 M β-mercaptoethanol) for a final concentration of 0.1 %
SDS, 50 mM β-mercaptoethanol. The tubes were heated at 100˚C for 5 minutes. The denatured
glycoproteins were allowed to cool to room temperature and 2.5 μL 15% NP-40 in water was added
18
(final concentration 0.75%). 1 μl of enzyme solution was added (2.5 mU enzyme) and the reaction was
incubated at 37˚C for 3 hours. Glycan release was confirmed by SDS-PAGE. Excess detergent, buffer
salts, and the core proteins were removed by porous graphitized carbon chromatography (SuperSil,
Thermo Fisher Scientific, Waltham, MA). Glycans were bound to a pre-equilibrated column in water
containing 0.1% TFA. The column was washed three times with water/0.1 %TFA (1ml), the glycans
were then eluted with 30% acetonitrile containing 0.1% TFA.
The glycan containing fractions were derivatized with d0-, d4-, d8- or d12-3 previously described
method. Briefly, N-linked samples were dried in the presence of the desired label (in 10% MeOH). The
dried sample was dissolved in 10 μL of a reaction reagent DMSO/glacial acetic acid (7:3) containing
1.0 M sodium cyanoborohydride. The glycan solutions were vortexed then centrifuged and incubated at
65°C for 3 h. Samples were allowed to cool to room temperature and 200 μl of ddH2O was added.
Excess tag was removed by a combination of solvent extraction and spin column chromatography. 200
μl portions of chloroform were added and the solution was allowed to partition, the chloroform layer
was then removed, this step was repeated two times. The resulting aqueous solution was added to a preequilibrated (according to manufacturer’s protocol) C18 MacroSpin column (Nest Group,
Southborough, MA), the columns were washed with 250 μl water (3x), the tagged glycans were eluted
in 5% methanol/water, and excess tag was eluted in 10% methanol/water fractions. The samples were
dried in preparation for analysis.
Samples were analyzed off-line using nanospray introduction via a standard electrospray ion source
into a QSTAR Pulsar i quadrupole orthoganol time-of-flight mass spectrometer (Applied
Biosystems/MDS-Sciex, Toronto, Cananda). Samples were dissolved in water, then diluted in an
isopropanol:water (1:9) solution containing 0.1% formic acid to a final concentration of 1 pmol/μl.
Aliquots of 5 μl were sprayed using nanospray tips with a 1 μm orifice pulled in-house using a capillary
puller (Sutter Instrument P80/PC micropipette puller, San Rafael, CA). An ionization potential of -1150
V produced a stable signal. All scans were acquired in the negative ion mode, and all spectra were
19
calibrated externally. The collision energy for tandem work involving CS tetramers were set to a level
where the precursor ion remained the most abundant signal in the spectrum, the best results were
obtained with a collision energy of -20 V, CAD gas 2. The MS/MS scans were summed for the -2
charge state for the CS oligosaccharides; this corresponded to one negative charge per sulfate group.
Abbreviations
ACN, Acetonitrile
CID, collision-induced dissociation
CS, chondroitin sulfate
CSA, chondroitin sulfate type A
CSB, chondroitin sulfate type B
CSC, chondroitin sulfate type C
ddH2O, MilliQ water (18MΩ)
DIEA, diisopropylethyl amine
DMF, Dimethyl formamide
dp, degree of polymerization
EDC, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride
ESI, electrospray ionization
Fmoc, 9-Fluorenylmethoxycarbonyl
HBTU, (O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate)
HOBt, 1-Hydroxybenzotriazole
LC liquid chromatography
MC, Methylene Chloride
MeOH, methanol
MS, mass spectrometry
PG, proteoglycan
SEC, size-exclusion chromatography
20
TIPS, Triisopropyl silane
TFA, Trifluoracetic acid
21
Scheme 1
+4
Glycan
+4
+4
Schematic of tag each module can be +0 or +4 daltons
+0
Healthy Tissue
Intensity
Extract and label glycans
Combine
m/z
Diseased Tissue
+8
MS2
Diseased Tissue
+12
Intensity
+4
Diseased Tissue
m/z
NH2
NH2
O
1
5
OH
O
O
N
H
O
N
H
O
N
H
NH2
O
NH2
O
O
2
N
H
NH2
H
N
H2N
N
H
O
H2N
O
NH2
O
H
N
N
H
NH2
N
H
O
NH2
O
H
N
O
3
4
6
H
N
O
O
O
O
N
H
NH2
N
H
H2N
O
O
N
H
7
8
O
H2N
H
N
N
H
O
22
Scheme 2
1.) deprotect
2.) Fmoc-Ala
HBTU, DIEA, DMF
A.
Ala-Fmoc
Fmoc
N
H
3.) deprotect
4.) Fmoc-Ala
HBTU, DIEA, DMF
H
N
1.) deprotect
2.) Boc-Anthranilic
Acid
HBTU, DIEA, DMF NH2 O
O
N
H
O
H
N
N
H
O
O
H
N
NH2
N
H
O
O
3.) Cleave from resin
4.) HPLC purify
H 2N
B.
O
O
O
O
+ R-NH2
CH3CN
Heat
O
H 2N
EDC,CH2Cl2
O
N
H
NHR
O
NH2
NH2 O
R-NH2=
NH2
NHR
HO
NH2
NH2
23
Fig 1
600
60
B.
A.
20
5
6
7
8
Compound
C.
NH2
NH2
O
1
5
OH
O
O
N
H
O
N
H
C3
10
B3
4
TAG
30
C2
3
C2
B2
2
H
N
NHAc
B3
C3
B2
40
0
1
O
OH
B1
OH
OH
O
HO
Y3
0
NHAc
OH
M-HSO3
100
Y1
OSO3
HOOC
O
HO
Y2
200
Y2
OH
O
(0,2)X
4
300
50
X4
O
Y1
400
OSO3
0,2
O
B1
Percent Total Ion Abundance
Absolute Intensity
500
Y3
HOOC
O
N
H
NH2
O
NH2
O
O
2
N
H
NH2
H
N
H2N
N
H
O
H2N
O
NH2
O
H
N
N
H
NH2
N
H
O
NH2
O
H
N
O
3
4
6
H
N
O
O
N
H
7
O
O
O
N
H
NH2
N
H
H2N
O
8
O
H2N
H
N
N
H
O
24
Fig. 2
Absorbance (AU, 232 nm)
0.30
A.
dp2
0.25
0.20
0.15
dp4
0.10
dp6
0.05
0.00
22
24
26
28
30
32
34
36
38
40
Min.
420.15
60
40%
B.
30%
40
418.81
30
20%
417.47
20
10%
10
416.13
0
416
418
420
422
m/z
60
Percent abundance
Intensity, counts
50
50
dp4 232 nm
C.
dp4 tag MS
40
30
20
10
0
10
20
30
40
Lyase digestion (% completion)
25
Intensity, counts
8.0
6.0
4.0
2.0
800
1000
10.0
978
980
6.0
14.0
0.30
0.20
0.10
1200
0.40
Bi(NeuAc)22-
1073.74
1072.40
1071.06
Tri(NeuAc)33-
1282.99
976
1069.72
976.71
975.37
14.0
1280.98
974
1278.97
972
1276.96
0.20
Bi(NeuAc)221283.01
0.60
973.02
0.80
Tri(NeuAc)431171.10
0.40
972.71
877.82
Tri(NeuAc)23-
1074.08
880
Tri(NeuAc)33-
878
Tri(NeuAc)23976.71
Tri(NeuAc)44-
12.0
878.08
10.0
876
Bi(NeuAc)23-
8.0
876.82
Tri(NeuAc)44-
855.00
874
805.31
2.0
875.81
6.0
Tri(NeuAc)34-
4.0
874.81
Fig 3 A
2.0
1070 1072 1074 1076
1276 1278 1280 1282 1284
1400 m/z
26
B
30
d0-100pmol
d4-200pmol
d8-300 pmol
d12-400 pmol
% Total Abundance
25
20
2.0
15
1.5
1.0
10
0.5
0.0
5
Bi(NeuAc)2 Tri(NeuAc)2
0
Bi(NeuAc)2
Tri(NeuAc)2
Tri(NeuAc)3
Tri(NeuAc)4
27
28
29
Table 1
Percentage
CSA
Percentage
CSB
Percentage
CSC
1:1:0
49.9 ± 1.1
46.0 ± 3.8
4.1 ± 2.8
1:0:1
54.0 ± 1.5
2.6 ± 2.0
43.4± 2.5
0:1:1
3.1 ± 1.7
50.0 ± 2.6
46.8 ± 2.1
1:1:1
32.0 ± 2.4
30.5 ± 4.4
37.3 ± 3.1
2:1:1
54.7 ± 4.0
23.3 ± 4.2
21.9 ± 3.2
1:2:1
24.1 ± 5.1
49.4 ± 2.9
26.5 ± 3.2
A. Mixture
CSA:CSB:CSC
26.6 ± 5.1
24.5 ± 5.4
48.9 ± 3.6
B. Digestion with
Chondroitinase
ABC
Digestion
Completion
Percentage
CSA
Percentage
CSB
Percentage
CSC
1:1:1 A:B:C
10%
62.8 ± 2.2
13.3 ± 1.5
24.1 ± 0.6
1:1:1 A:B:C
20%
59.0 ± 2.1
22.2 ± 1.4
18.8 ± 1.2
1:1:1 A:B:C
30%
30.7 ± 3.4
29.2 ± 4.7
40.1 ± 4.1
1:1:1 A:B:C
40%
34.8 ± 4.5
26.3 ± 2.4
38.9 ± 3.1
1:1:2
30
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