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`Author Manuscript
`J Am Soc Mass Spectrom. Author manuscript; available in PMC 2009 January 1.
`Published in final edited form as:
`J Am Soc Mass Spectrom. 2008 January ; 19(1): 82–90.
`
`Tandem MS can Distinguish Hyaluronic Acid from N-
`Acetylheparosan
`
`Zhenqing Zhang1,#, Jin Xie1,#, Jian Liu2, and Robert J. Linhardt1,*
`1Departments of Chemistry and Chemical Biology, Chemical and Biological Engineering and Biology, Center
`for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, 12180
`2Division of Medicinal Chemistry and Natural Products, School of Pharmacy University of North Carolina,
`Chapel Hill, North Carolina 27599
`
`Abstract
`Isobaric oligosaccharides enzymatically prepared from hyaluronic acid (HA) and N-acetylheparosan
`(NAH), were distinguished using tandem mass spectrometry. The only difference between the two
`series of oligosaccharides was the linkage pattern (in HA 1→3 and in NAH 1→4) between glucuronic
`acid and N-acetylglucosamine residues. Tandem mass spectrometry afforded spectra, in which
`glycosidic cleavage fragment ions were observed for both HA and NAH oligosaccharides. Cross-
`ring cleavage ions 0,2An and 0,2An-h (n is even number) were observed only in GlcNAc residues of
`NAH oligosaccharides. One exception was an 0,2A2 ion fragment observed for the disaccharide from
`HA. These cross-ring cleavage fragment ions are useful to definitively distinguish HA and NAH
`oligosaccharides.
`
`Keywords
`hyaluronic acid; N-acetylheparosan; oligosaccharide; tandem mass spectrometry; fragmentation
`
`Introduction
`Glycosaminoglycans (GAGs) are linear, acidic polysaccharides found on cell surfaces and in
`the surrounding extracellular matrix. GAGs participate in and regulate many cellular events
`and physiological and pathophysiological processes, such as cell proliferation and
`differentiation, cell-cell and cell-matrix interactions, viral infection through their interaction
`with different proteins [1-3]. GAGs are divided into four main categories: hyaluronic acid, or
`hyaluronan (HA), chondroitin/dermatan sulfate, N-acetylheparosan (NAH)/heparan sulfate/
`heparin, and keratan sulfate, based on their monosaccharide composition and the configuration
`and position of the glycosidic bonds between these monosaccharides. The specificity of the
`interactions between GAGs and proteins results from structural diversity of GAGs defined by
`their size, saccharide composition and sequence, charge density [4,5]. Thus, understanding the
`structure of a GAG is essential in understanding its activity and biological functions. HA and
`NAH are biosynthesized by both prokaryotic and eukaryotic cells. HA is co-polymer linked
`polymer of β-1,4 D-glucuronic acid (GlcA) and β-1,3 D-N-acetylglucosamine (GlcNAc). [6]
`
`* Corresponding author, Phone: 518-276-3404, Fax: 518-276-3405, Email: Linhar@rpi.edu.
`#These authors contributed equally to this work
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`NAH is a co-polymer of β-1,4 GlcA and α-1,4 GlcNAc and the biosynthetic precursor of the
`mammalian GAGs, heparin and heparan sulfate [7]. HA can be prepared by bacterial
`fermentation of Streptococcus zooepidemicus [8], or enzymatically using biosynthetic enzymes
`prepared from Pasturella multocida [9]. Bacteria use HA as camouflage to enhance their ability
`to infect higher animals [10]. Similarly, Escherichia coli strain K5 produces capsular
`polysaccharide comprised of NAH [11]. NAH is also important as it represents a considerable
`portion of the sequence of mammalian heparan sulfate [12,13]. HA and NAH are the two
`simplest GAGs since neither have sulfo groups and, thus, consist of only a single sequence
`(Figure 1). HA and NAH have very similar structures, differing only in the position of their
`glycosidic linkage between GlcA and GlcNAc. This difference in structure results from very
`different biosynthesis pathways for HA and NAH [14-17] and gives each polysaccharide
`unique biological functions and distinctive structure-activity relationships [18].
`
`The oligosaccharides prepared from GAGs, by controlled enzymatic depolymerization, are
`often studied to define the minimum structural requirements for biological activity [19,20].
`ESI-MS is particularly useful to monitor these GAG-derived oligosaccharides due to its soft
`ionization [21,22]. LC-ESI-MS, relying on reversed-phase ion-pairing (RPIP)-high
`performance liquid chromatography (HPLC), has employed volatile ion-pairing reagents [23,
`24] to successfully analyze HA and NAH [25]. Unfortunately, this method results in nearly
`identical retention times for the HA and NAH oligosaccharides and since they have identical
`masses, it is not possible to differentiate these oligosaccharides. Most laboratories routinely
`rely on nuclear magnetic resonance (NMR) spectroscopy, requiring relatively large amounts
`of samples, to distinguish HA and NAH oligosaccharides [26,27]. These two series of
`oligosaccharides can also be differentiated based on their susceptibilities to specific
`polysaccharide degrading enzymes, where the degradation can be detected by ultraviolet
`spectroscopy (UV), HPLC, thin layer chromatography (TLC), polyacrylamide gel
`electrophoresis or change in viscosity. A new method, which could confirm the identity of
`polysaccharide made by small samples of new isolates containing both HA and NAH, would
`be useful.
`
`Tandem mass spectrometry offers a sensitive and more readily available alternative to NMR
`for the analysis of GAG oligosaccharides. ESI tandem MS has been previously used to analyze
`HA oligosaccharides and glycosidic fragment ions were previously observed with single or
`multiple charges [28,29]. Unfortunately, these fragment ions would be expected to form from
`both HA and NAH oligosaccharides.
`
`This article describes a method for distinguishing HA and NAH oligosaccharide mixtures
`prepared by partial enzymatic digestion. RPIP-LC-ESI-MS, and tandem MS are applied to
`analyze these oligosaccharides and distinguish HA and NAH oligosaccharides based on the
`relative abundance of their characteristic fragment ions.
`
`Materials and Methods
`Preparation of oligosaccharides mixture
`N-acetylheparosan (NAH) was isolated through fermentation of E. coli K5 and purified as
`previously described [11]. The NAH 500 μg/50 μl was incubated in 50 mM sodium phosphate
`buffer pH 7.0 with heparin lyase III (10 m-units, Sigma Chemicals, St Louis, MO, U.S.A.) at
`37°C for 10 h with aliquots removed at various time points for analysis. The aliquots were
`heated in a boiling water bath for 10 min. to halt the reaction. The denatured protein was
`removed by centrifugation at 12000 × g for 10 min. Hyaluronic acid (HA, from rooster comb)
`was purchased from Sigma. Chondroitin lyase ABC (5 m-units, Seikagaku Biochemical,
`Tokyo, Japan) was used to digest HA (500 μg/50 μl) in 50 mM sodium phosphate buffer pH
`7.0 at 37°C for 10 h and aliquots were removed periodically and collected as described above.
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`TLC conditions
`The progress of the enzymatic depolymerization was monitored by measuring the HA and
`NAH oligosaccharides present in aliquots taken throughout the reaction by TLC on a precoated
`silica gel-60 aluminum plates (Merck, Darmstadt, Germany) (1 × 5 cm) or (10 cm × 5 cm)
`eluted with a solvent system consisting of n-butanol/formic acid/water, 4:8:1 (v/v). The eluted
`plate was stained by dipping in a reagent containing 1 ml of 37.5% HCl, 2 ml of aniline, 10 ml
`of 85% H3PO3, 100 ml of ethyl acetate and 2 g diphenylamine) for 3 sec and heating at 150 °
`C for 10 sec. [30].
`
`LC-MS/MS
`
`LC MS analyses were performed on Agilent 1100 LC/MSD instrument (Agilent Technologies,
`Inc. Wilmington, DE, U.S.A.) equipped with an ion trap, binary pump and a UV detector. The
`column was a 5 μm Agilent Zorbax SB-C18 (0.5 × 250 mm) from Agilent Technologies. Eluent
`A was water/acetonitrile (85:15), v/v and eluent B was water/acetonitrile (35:65) v/v. Both
`eluents contained 12 mM tributylamine (TBA) and 38 mM NH4OAc and their pH was adjusted
`to 6.5 with HOAc. The product mixtures of the HA and NAH enzyme digestions were each
`diluted 10-fold with water and 1 μl of the resulting analytes were individually injected by auto-
`sampler. A gradient of 0% B for 15 min, and 0-100% B over 85 min. was used at a flow rate
`of 10 μl/min. Mass spectra were obtained using an Agilent 1100 series Classic G2445D LC/
`MSD trap (Agilent Technologies, Inc. Wilmington, DE, U.S.A.). The electrospray interface
`was set in negative ionization mode with the skimmer potential -40.0 V, capillary exit -120 .5
`V and a source temperature of 325 °C to obtain maximum abundance of the ions in a full scan
`spectra (150–1500 Da, 10 full scans/s). Nitrogen was used as a drying (5 liters/min) and
`nebulizing gas (20 p.s.i.). Auto MS/MS was turned on in these experiments using an estimated
`cycle time of 0.07 min. Total ion chromatograms (TIC) and mass spectra were processed using
`Data Analysis 2.0 (Bruker software).
`
`Results and Discussion
`Chondroitin lyase ABC was used to digest HA and heparin lyase III was used to digest NAH
`and both digestions were carried out to ~30% completion. The extent of digestion, reflected in
`the value “m” (at 100 % digestion m = 0) was monitored by TLC (Figure 2A). The
`oligosaccharides resulting from HA had the general structure ΔUA (1[→3) GlcNAc (1→4)
`GlcA (1→3]m) GlcNAc (ΔUA, 4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid, m =
`0,1,2…). The oligosaccharides resulting from NAH had the general structure ΔUA (1[→4)
`GlcNAc (1→4) GlcA (1→4]m) GlcNAc.
`Next, LC-ESI-MS analysis was performed on HA and NAH oligosaccharides obtained at 30%
`reaction completion (Figure 2B and C). Peaks corresponding to a degree of polymerization
`(dp) from 2 to 20 were observed in the mass spectra of both HA and NAH oligosaccharides
`(Table 1). The ESI-MS spectra of HA and NAH oligosaccharides were identical across a range
`of dp values. For example, the largest oligosaccharides from HA and NAH (dp 20) afforded a
`calculated mass of 3791.1 based on the fully protonated acidic form. The mass spectra of both
`showed identical multiply charged molecular-ion peaks [M-5H]5- and [M-4H]4- at m/z 757.3
`and 946.9, respectively (Figure 3), confirming a length of 20 saccharide units.
`
`Tandem mass spectrometry can result in both glycosidic linkage and cross-ring cleavages. All
`product ions are labeled according to the Domon and Costello nomenclature. [31] The cross-
`ring cleavages hold the key in distinguishing HA and NAH oligosaccharides. Tandem mass
`spectra of the smallest products, the two disaccharides (dp2) from HA and NAH, were
`examined to see if they could be distinguished (Figure 2A and B). Tandem MS, on the singly-
`charged precursor at m/z 378.1, afforded a cross-ring cleavage ion fragment 0,2A2 at m/z 276.9
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`in both the HA and NAH disaccharides but also a unique ion fragment 0,2A2-h at m/z 258.9 in
`the spectrum of NAH disaccharide, corresponding to a loss of H2O from 0,2A2 (Figure 4).
`The tandem mass spectra of two tetrasaccharides (dp4) were next examined (Figure 5 A and
`B) to determine whether this unique fragment could be used to distinguish oligosaccharides
`derived HA and NAH. Glycosidic cleavage fragment ions were observed, from the singly
`charged the precursors molecular ions at m/z 757.1, for both HA and NAH tetrasaccharides.
`Again, the cross-ring cleavage ion fragments 0,2A2-h m/z 258.9 and 0,2A4-h m/z 638.1 were
`observed only in the NAH. When the tandem MS was performed on the doubly charged
`molecular ions of the two hexasaccharides (dp6) at m/z 576.8 (Figure 6A and B), the fragments
`observed were similar to those observed in the tandem mass spectra of the dp 4 [M-H]-
`precursor. In addition to the glycosidic cleavage ion fragments, observed in tandem mass
`spectra of both HA and NAH hexasaccharides, A-type ion
`fragments 0,2A2, 0,2A4, 0,2A6, 0,2A2-h, 0,2A4-h and 0,2A6-h were also observed in the tandem
`mass spectrum of the NAH hexasaccharide. The 0,2A6 and 0,2A6-h fragments were mainly
`observed as doubly charged ions at m/z 517.1 and m/z 508.1, respectively. Cross-ring cleavage
`ion fragments were not observed in spectrum of HA hexasacchrides. In the spectra of HA
`tetrasaccharide and hexasaccharide, the relative intensities of fragment ions were different. In
`the spectrum of HA hexasaccharide, the C3 fragment ion at m/z 554.1 is of much lower intensity
`than the C3 fragment ion in the spectrum of HA tetrasaccharide (Figures 5A and 6A). The
`C4/Z4 fragment ion at m/z 757.2 dominates the spectrum of NAH hexasaccharide, while all
`the peaks in the spectrum of NAH tetrasaccharide are of similar relative intensities (Figure 5B
`and 6B). These differences between tetrasaccharide and hexasaccharides would appear to
`suggest that the charge state influences the product ion pattern. All ion fragments in the tandem
`mass spectra of dp2 to dp12, derived from HA and NAH, are presented in Table 2 and Table
`3, respectively. Tandem MS was performed on singly or multiply charged molecular ions
`(labeled with asterisks in Table 1). 0,2A-type ion fragments were observed in the GlcNAc
`residue of NAH oligosaccharides, but were not observed in the GlcNAc residues of HA
`oligosaccharides. The larger oligosaccharides of HA and NAH from dp14 to dp20 shown in
`the TIC (Figure 2) have a similar fragmentation to that of smaller oligosaccharides, dp2 to dp
`12, in which the difference between HA and NAH also could be observed (data not shown).
`Interestingly, only dp 2 of HA gave an 0,2A fragment (but not an 0,2A-h fragment),
`making 0,2A-h fragment a unique marker for NAH.
`
`Ion fragments, of the 0,2A-type observed in the NAH GlcNAc residues, containing 1→4 linkage
`suggest the fragmentation mechanism is a retro-aldol rearrangement of oligosaccharide ions
`in the negative mode [32-35]. This mechanism requires an open-ring reducing terminal
`aldehyde, and such structures are formed by C-type fragmentation [34-38]. The 0,2An (n=even
`number) ions formed from cross-ring cleavage to the internal glucosamine residues are
`therefore likely to arise from the Cn (n=even number) ions by retro-aldol rearrangement. These
`A-type fragment ions are observed in residues that are 4- or 6- linked [21,34,36-42].
`Furthermore, in NAH oligosaccharides, the free hydroxy group at position 3 can accept a proton
`from position 4 after cleavage of the GlcNAc ring by 0,2A type fragmentation (Figure 7)
`affording water and the negatively charged 0,2An-h fragment. These dehydrated A-type
`fragment ions also are observed with the residue requiring an unsubstituted hydroxyl in the 3-
`position in other oligosaccharides [34,36,39,43]. In the HA oligosaccharides, the bonds at C-3
`of GlcNAc are substituted perhaps making these more stable than NAH oligosaccharides. The
`exception to this rule is dp2 from HA, where the 0,2A2 ion fragment is observed. This may
`result from the small size of the dp 2 molecule, preventing it from dissipating vibrational
`energy, resulting in its fragmentation. Despite its small size, the HA disaccharide still fails to
`afford a 0,2An-h fragment ion because of its substitution at position 3. Here, the presence
`of 0,2A-type fragment ions can be used as a diagnostic tool to distinguish HA disaccharide from
`longer HA oligosaccharides, in cases where the charge state is unknown.
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`In conclusion, the current study clearly demonstrates LC-MS/MS can be used to differentiate
`HA and NAH oligosaccharides. The sensitivity of this method is better than 100 ng for each
`oligosaccharide separated using RPIP-HPLC. A-type fragment ions from GlcNAc residues
`were shown as diagnostic peaks in MS/MS spectra of NAH oligosaccharides, but not in the
`spectra of HA oligosaccharides. One exception was an 0,2A2 ion fragment observed for the
`disaccharide from HA. This approach should be useful for screening the capsules of bacterial
`isolates to determine whether they are composed of HA or NAH polysaccharides. This method
`might also be useful in distinguishing between small quantities of HA and NAH derived
`oligosaccharides isolated from animal tissues. This method should also be useful as a
`complement approach to confirm the structure of unsulfated chondrointin oligosaccharides that
`have the same linkage pattern as HA, containing 3-linked HexNAc residues.
`
`Acknowledgements
`The authors thank Tatiana Laremore for her careful reading of this manuscript and the NIH for supporting this research
`through grants GM38060 and HL62244.
`
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`37. Pfenninger A, Karas M, Finke B, Stahl B. Structural analysis of underivatized neutral human milk
`oligosaccharides in the negative ion mode by nano-electrospray MS(n) (part 1: methodology). J Am
`Soc Mass Spectrom 2002;13:1331–1340. [PubMed: 12443024]
`38. Pfenninger A, Karas M, Finke B, Stahl B. Structural analysis of underivatized neutral human milk
`oligosaccharides in the negative ion mode by nano-electrospray MS(n) (part 2: application to isomeric
`mixtures). J Am Soc Mass Spectrom 2002;13:1341–1348. [PubMed: 12443025]
`39. Chai W, Piskarev V, Lawson AM. Branching pattern and sequence analysis of underivatized
`oligosaccharides by combined MS/MS of singly and doubly charged molecular ions in negative-ion
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`Figure 1.
`Structure of HA and NAH.
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`NIH-PA Author Manuscript
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`NIH-PA Author Manuscript
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`Figure 2.
`Total ion chromatogram (TIC) of HA oligosaccharides (A). TLC analysis of NAH
`oligosaccharides (B). TIC of NAH oligosaccharides (C).
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`NIH-PA Author Manuscript
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`NIH-PA Author Manuscript
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`Figure 3.
`ESI mass spectra of dp20 of HA (A) and NAH (B).
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`NIH-PA Author Manuscript
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`Figure 4.
`MS/MS spectra of disaccharides (dp2) from HA (A) and NAH (B).
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`Figure 5.
`MS/MS spectra of tetrasaccharides (dp4) from HA (A) and NAH (B).
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`Figure 6.
`MS/MS spectra of hexasaccharides (dp6) from HA (A) and NAH (B).
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`757.3
`681.5
`
`947.2*
`852.2*
`757.4*
`
`1136.9
`1010.3
`883.8*
`757.3*
`630.9
`
`1326.8
`1136.8
`946.8*
`757.2*
`567.8*
`
`[M-5H]5-
`
`[M-4H]4-
`
`[M-3H]3-
`
`[M-2H]2-
`
`1136.3
`757.1*
`378.1*
`
`[M-H]-
`
`757.2
`
`[2M-H]-
`
`Fragments marked were used as precursor ions in tandem MS.
`
`*
`
`3791.1
`3412.0
`3032.9
`2653.8
`2274.7
`1895.5
`1516.4
`1137.3
`758.2
`379.1
`
`Calculated mass
`
`dp20
`dp18
`dp16
`dp14
`dp12
`dp10
`dp8
`dp6
`dp4
`dp2
`
`NIH-PA Author Manuscript
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`Major molecular ions observed in the ESI mass spectra of HA and NAH oligosaccharides.
`NIH-PA Author Manuscript
`NIH-PA Author Manuscript
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`Table 1
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`775.2
`978.3
`1154.3
`1375.4
`
`757.2
`933.2
`1136.3
`1312.3
`757.1
`845.6
`947.2
`1035.5
`915.2
`1118.3
`1294.3
`1497.3
`
`775.2
`978.3
`1154.3
`1375.4
`
`757.2
`933.2
`1136.3
`1312.3
`757.2
`845.5
`
`739.1
`915.2
`1118.3
`1294.3
`1497.5
`
`599.1
`775.1
`978.3
`1154.3
`
`554.1
`757.1
`933.2
`1136.3
`1312.3/655.7
`
`536.8
`739.1
`915.2
`
`396.0
`599.1
`775.1
`978.2
`
`378.0
`554.1
`757..2
`933.2/466.1
`
`739.2
`915.2
`
`396.0
`599.1
`
`378.0
`554.1
`
`359.9
`536.1
`
`dp12
`
`dp10
`
`dp8
`
`dp6
`
`dp4
`
`276.9
`
`dp2
`
`dp =2 n
`
`*
`
`Yn-8
`Yn-7
`Yn-6
`Yn-5
`Yn-4
`Yn-3
`Yn-2
`Yn-1
`Cn-8/Zn-8
`Cn-7
`Cn-6/Zn-6
`Cn-5
`Cn-4/Zn-4
`Cn-3
`Cn-2/Zn-2
`Cn-1
`Bn-7
`Bn-6
`Bn-5
`Bn-4
`Bn-3
`Bn-2
`Bn-1
`0,2An-h
`0,2An *
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`Fragment ions observed in the product-ion spectra of HA oligosaccharides
`NIH-PA Author Manuscript
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`Table 2
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`775.2
`978.3
`1154.3
`1375.4
`
`757.2
`933.2
`1136.3
`1312.3/656.1
`757.1
`845.8
`947.2
`
`536.1
`739.1
`915.2
`1118.2
`1294.3
`1497.4
`
`258.8
`276.8
`638.1
`656.1
`1017.1/508.1
`1035.1/517.1
`1396.4/697.7
`1414.4/706.6
`887.7
`896.7
`717.7
`723.7
`
`dp12
`
`775.2
`978.3
`1154.3
`1375.4
`
`757.2
`933.2
`1136.3
`1312.3/656.2
`757.2
`845.8
`
`536.1
`739.1
`915.2
`1118.3
`1294.3/646.2
`1497.4/748.8
`
`258.8
`276.8
`638.1
`656.1
`1017.1/508.1
`1035.1/517.1
`1396.4/697.7
`1414.4/706.6
`887.3
`8