2Mass Spectrometry Facility, Department of Psychiatry, School of Medicine, The University of North Carolina, Chapel Hill, NC 27599, USA, 3Department of Chemistry, The University of North Carolina, Chapel Hill, NC 27599, USA, and 4Division of Medicinal Chemistry and Natural Products, School of Pharmacy, The University of North Carolina, Chapel Hill, NC 27599, USA
Received on December 18, 2000; revised on February 5, 2001; accepted on February 5, 2001.
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Abstract |
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Key words: heparan sulfate/oligosaccharides/nano-electrospray/mass spectrometry/antithrombin
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Introduction |
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The diversified biological functions of HS are likely attributed to their unique sulfated saccharide sequences. Although the detailed mechanism for regulating the biosynthesis of HS with a defined saccharide sequence is unknown, it has been speculated to be related to the presence of various isoforms of each class of HS biosynthetic enzyme.(Liu et al., 1999b) Indeed, HS N-deacetylase/N-sulfotransferase, 3-O-sulfotransferase, and 6-O-sulfotransferase are present in multiple isoforms, and each isoform is believed to recognize the saccharide sequence around the modification site to generate a specific sulfated saccharide sequence (Ishihara et al., 1993
; Cheung et al., 1996
; Aikawa and Esko, 1999
; Liu et al., 1999b
; Habuchi et al., 2000
; Aikawa et al., 2001
). This activity regulates the concentration and distribution of specific recognition motifs on the cell surface. For example, HS modified by 3-O-sulfotransferase isoform 1 (3-OST-1) binds to antithrombin (AT) with anticoagulant activity (Liu et al., 1996
), whereas the HS modified by 3-O-sulfotransferase isoform 3 (3-OST-3) binds to herpes simplex 1 envelope glycoprotein D to serve as an entry receptor for herpes simplex virus 1 infection (Shukla et al., 1999
).
Heparin is the most commonly used anticoagulant drug. Heparin and HS have very similar disaccharide compositions, except that heparin has a greater content of iduronic acid and a higher number of sulfates per polysaccharide chain (Lindahl et al., 1998). The HS- and heparin-involved anticoagulation mechanisms have been studied extensively. It is now known that HS and heparin interact with AT, a serine protease inhibitor, to inhibit the activities of thrombin and factor Xa in the blood coagulation cascade (Rosenberg et al., 1997
). Anticoagulant-active HS (HSact) and heparin contain one or multiple AT binding sites per polysaccharide chain. This binding site is a pentasaccharide with a structure of -GlcNS(or Ac)6S-GlcA-GlcNS3S(±6S)-IdoA2S-GlcNS6S- (where GlcN is glucosamine, IdoA is iduronic acid, GlcA is glucuronic acid, S is sulfate, and Ac is acetate). The 3-O-sulfation of glucosamine to generate GlcNS3S(6S) is a critical modification that results in the formation of HSact. HS 3-O-sulfotransferase 1 (3-OST-1) (EC 2.8.2.23) is the critical enzyme that forms the AT binding site (Liu et al., 1996
; Shworak et al., 1997
).
Because there are few techniques to separate and characterize HS oligosaccharides, the relationship between the saccharide sequences and diversified biological function remains obscure. Despite the recent progress in purifying and sequencing HS oligosaccharides, the available techniques for completely characterizing HS structures are still inadequate. It is important to note that a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) technique was developed to analyze HS oligosaccharides (Juhasz and Biemann, 1994; Rhomberg et al., 1998
). This approach affords high sensitivity and enhanced throughput (Venkataraman et al., 1999
; Shriver et al., 2000
). The HS oligosaccharides, however, are not observed directly. Instead, this method necessitates formation of a complex between the HS and a basic peptide prior to analysis. Hence, direct structural information via tandem MS is not available; only the mass difference between the complex and free peptide is observed. Moreover, quantitative analysis has not been reported with this method.
Electrospray ionization mass spectrometry (ESI-MS) techniques have attracted attention for analysis of HS and chondroitin sulfate oligosaccharides because they provide high mass accuracy, structural information, and the ability to quantify the analyte (Chai et al., 1998; Kim et al., 1998
; Desaire and Leary, 2000; Yang et al., 2000
). Those reported approaches utilize microscale, forced-flow ESI, at flow rates of 520 µl/min and sample concentrations of 5200 µM. Thus, each analysis consumes about 40400 pmole of oligosaccharide. Unfortunately, the application of ESI-MS for analyzing biologically active HS oligosaccharides has been hindered due to high sample consumption.
Nano-electrospray ionization mass spectrometry (nESI-MS) has shown promise for analyzing proteins and other biomolecules because of its reduced flow rate and sample consumption (Wilm and Mann, 1996; Bahr et al., 1997
). These advantages make it a choice method for the study of peptides in the low fmole range. Therefore, we have adapted nESI-MS for HS analyses with high sensitivity. In this manuscript, we report that nESI-MS was used to analyze HS pentasaccharides at a concentration as low as 50 nM. Based on our estimation, this approach consumes 330 fmoles (x 1015 mole) to obtain a spectrum with a signal-to-noise ratio greater than 5. In addition, we found a linear relationship between the relative response of the molecular ion and the concentration of the analyzed 3-OH pentasaccharide, suggesting that nESI-MS can be utilized as a sensitive approach to quantify HS oligosaccharides. We used nESI-MS to quantify a concentration of 3-O-sulfated pentasaccharide that was prepared from enzymatic modification. The result is very similar to the estimation from [35S]radioactivity.
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Results |
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Analysis of 3-O-sulfated pentasaccharide
We decided to test our method by quantifying a sample of biologically active HS oligosaccharides, prepared from an enzymatic modification of the 3-OH pentasaccharide. The AT-binding pentasaccharide was selected in this experiment for the following reasons. First, we are capable of radiolabeling the AT-binding pentasaccharide with [35S] (3-O-sulfated pentasaccharide). Therefore, the amount of the pentasaccharide is easily estimated based on the specific [35S] radioactivity. This information was used to validate the result from a quantitative analysis by nESI-MS as described below. Second, the purification of the AT-binding pentasaccharide can be easily carried out. The binding affinity of the 3-OH pentasaccharide (non-AT-binding pentasaccharide) to AT is about 16,000-fold less than that of 3-O-sulfated pentasaccharide (AT-binding pentasaccharide) (Atha et al., 1985). Using AT-affinity chromatography combined with anion exchange-HPLC, the 3-OH and 3-O-sulfated pentasaccharides are readily separated.
The 3-O-sulfated pentasaccharide was prepared by incubating purified HS 3-O-sulfotransferase 1 and 3-OH pentasaccharide in the presence of [35S]PAPS as described under Materials and methods. The 3-O-sulfated pentasaccharide was purified by using an AT-affinity column followed by silica-based polyamine high-performance liquid chromatography (PAMN-HPLC). We confirmed that 3-O-sulfated pentasaccharide binds to AT by using affinity coeletrophoresis (data not shown).
The nESI-MS spectrum of the 3-O-sulfated pentasaccharide is shown in Figure 5. (We calculated the molecular weight of 3-O-[35S]sulfated pentasaccharide based on 32S, because [35S]sulfate represents less than 0.4% of total 3-O-sulfate.) The pentasaccharide ion current is partitioned between triply charged and quadruply charged ions [M-nH]n at m/z 501 and 376, respectively. The deconvoluted spectrum is also shown in Figure 5, inset A. The signals at molecular mass of 1508, 1530, and 1546 correspond to the 3-O-sulfated pentasaccharide, and its sodium and potassium adducts. (The sodium and potassium adducts of 3-O-sulfated pentasaccharide were observed during the analysis by nESI-MS. However, we did not observe such high levels of salt adducts to 3-OH pentasaccharide [Figure 4A]. We believe the reason for the presence of sodium and potassium adducts in 3-O-sulfated pentasaccharide is likely due to incomplete desalting after the purification by PAMN-HPLC, which was eluted by 1 M potassium phosphate monobasic.) It should be noted that we did not observe 3-OH pentasaccharide signals at m/z 475 and 356, suggesting that our preparation of 3-O-sulfated pentasaccharide is pure and desulfation in the source is negligible.
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To conduct a quantitative analysis by nESI-MS, the 3-O-sulfated pentasaccharide was mixed with the internal standard, UA-GlcNAc, and analyzed by nESI-MS (spectra not shown). Based on the relative intensity of the signals of 3-O-sulfated pentasaccharide and
UA-GlcNAc, the calculated concentration of analyzed 3-O-sulfated pentasaccharide is 0.68 ± 0.03 µM (the data point is represented as a filled-in triangle in Figure 4B). This value is within 20% of 0.85 µM, the value estimated from specific [35S]radioactivity.
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Discussion |
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It should be noted that the conditions for nESI-MS are significantly milder than forced-flow ESI-MS and the ionization efficiency for nESI-MS is estimated to be 510 times higher than ESI-MS (Wilm and Mann, 1996). We used a low negative nESI voltage (530 to 630 V) and low cone voltage (13 V) during the analysis compared to ESI voltages used at higher flow (2000 to 4000 V and cone voltage at around 60 V). In negative ESI, higher voltages contribute to corona discharge and thus increased chemical noise. We have noted that milder nESI-MS conditions reduce desulfation of the oligosaccharides as well. Indeed, we observed a relatively low level of desulfation during the analyses of disaccharides and pentasaccharides, except for the trisulfated disaccharide (
UA2S-GlcNS6S, Figure 2F). We also observed an increase in desulfation of the pentasaccharides when higher cone voltages were employed for nESI-MS. In addition, we found that adding 0.2% of imidazole is helpful to reduce the level of sodium adducts of pentasaccharides and improve sensitivity (spectra not shown). It is known that imidazole and pyridine suppress the formation of sodium/oligonucleotide adducts (Grieg and Griffey, 1995
).
Like previously reported ESI-MS methods, nESI-MS is capable of quantifying HS oligosaccharides, provided that appropriate standards are available. Such a quantitative capability will play an important role in sequencing analysis of HS oligosaccharides because it is necessary to monitor the conversion yield during each sequencing step. The currently reported sequencing approaches for underivatized oligosaccharides utilize the absorbance at UV 232 nm or radioactivity (Merry et al., 1999; Venkataraman et al., 1999
). However, the sensitivity at 232 nm is relatively low. Furthermore, for those samples without radiolabels or absorbance at 232 nm, neither method can be used. Our data suggest that the standard curve constructed from the 3-OH pentasaccharide could be used to estimate the amount of the 3-O-sulfated pentasaccharide. It might be essential to choose appropriate standards to generate a suitable curve for quantitative analysis of an unknown structure. Ideally, the oligosaccharides with known structures would be used to generate standard curves which project the amount of oligosaccharides with unknown structures. It is unfortunate that we are presently unable to investigate the general rules for choosing standards for quantitative analysis because well-defined HS oligosaccharides are not available.
In summary, nESI-MS is a sensitive tool to quantitatively analyze HS oligosaccharides. This technique will provide an alternative approach to MALDI-MS for interrogating HS oligosaccharides. Each technique has its unique strengths for analyzing HS oligosaccharides. Combining MALDI-MS and nESI-MS will be essential to characterize the structures of unknown HS oligosaccharides with greater confidence.
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Materials and methods |
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nESI-MS analysis
A Micromass Quattro II with QHQ geometry and the Z-Spray source was used in these experiments. Borosilicate glass capillaries (OD 1 mm and ID 0.57 mm) were pulled on a glass capillary puller (Narishige Company, Japan). A titanium wire (99.99%, Goodfellow, Inc.) was inserted in the distal end of the glass capillary, to provide electrical contact to the solution. Negative nESI voltage was maintained between 530 V and 650 V during the analysis. To prevent desulfation in the gas phase, the absolute value of the sampling cone voltage was maintained < 13 V. Unit mass resolution was used in all MS1 scans. During MS/MS experiments, argon was used as the collision gas. Collision energy was kept low (12 eV), because desulfation is a facile gas phase process. Spectra were acquired over 300800 m/z, at 2 s per scan, 16 pts/Da.
In the neutral loss scans, MS/MS spectra were obtained by scanning Q1 and Q3 with an offset of 26.7 or 20 m/z in their scan cycles, corresponding to the loss of sulfate from the triply or quadruply charged pentasaccharides, respectively. To maximize the sensitivity of neutral loss detection, resolution was lowered so that peak widths were ca. 1.2 Da at full width at half maximum.
Quantitative analysis of HS pentasaccharides by nESI-MS
The 3-OH pentasaccharide was diluted with a solvent containing 0.2% NH4OH, 0.2% imidazole, and 50% acetonitrile. The internal standard, UA-GlcNAc, was spiked into each sample to yield a final concentration of 15 µM. The final 3-OH pentasaccharide concentration ranged from 0.05 µM to 10 µM. Approximately 2 µl of the mixture was loaded into a pulled-glass capillary and infused for 5 min. Ion chronograms were constructed and selected from 1 min of analysis time, during which time signal response was strongest. All peaks were integrated, and the ion intensities of the [M-3H]3 and [M-4H]4 peaks of the 3-OH pentasaccharide were summed, and divided by the intensity of the internal standard. Experiments were performed in duplicate, and the relative intensities were averaged together. These relative intensities were plotted against the concentration of the 3-OH pentasaccharide. To quantify the 3-O-sulfated pentasaccharide, the pentasaccharide (11,500 c.p.m./µl or 0.85 µM) was diluted with the solvent mixture as described above in the presence of 15 µM
UA-GlcNAc.
Preparation of 3-O-pentasaccharide
Purified 3-OST-1 (70 ng) was mixed with 1 µg of the chemically synthesized pentasaccharide and 10 µM [35S]PAPS (7.2 x 106 c.p.m., 14,000 c.p.m./pmole) in 50 µl of the enzyme reaction buffer containing 50 mM 2-[N-morphilino]ethanesulfonic acid, 1% Triton X-100, 1 mM MgCl2, 2 mM MnCl2, and 168 µg/ml of bovine serum albumin, pH 7. The reaction was incubated at 37°C for 2 h and was terminated by heating at 100°C for 2 min. The resultant was centrifuged at 14,000 r.p.m. for 1 min to remove insoluble matter. The 3-O-[35S]sulfated pentasaccharide was purified by an antithrombin-Con A affinity column as described previously (Liu et al., 1996). Briefly, the resulting supernatant was mixed with 50 µl water and then incubated with 50 µl of a buffer, containing final concentrations of 10 mM Tris, 150 mM NaCl, 1 mM Ca2+, 1 mM Mg2+, 1 mM Mn2+, and 0.1 mg/ml of antithrombin (pH 7.5), at room temperature for 30 min. The solution was then loaded onto a 300-µl ConA-Sepharose column equilibrated with a buffer containing 10 mM Tris, 0.0004% Triton X-100, and 150 mM NaCl, pH 7.5. The gel was incubated at room temperature for an additional 30 min, washed with 3 x 1ml of a buffer containing 10 mM Tris, 0.0004% Triton X-100, and 150 mM NaCl (pH 7.5). The 3-O-sulfated pentasaccharide was eluted from the gel by using 2 x 0.5 ml of a buffer containing 10 mM Tris, 1000 mM NaCl, and 0.0004% Triton X-100 (pH 7.5). The eluted material was dialyzed against 50 mM ammonium bicarbonate using 3,500 MWCO dialysis tubing (Spectrum). Ten reactions were prepared to obtain a sufficient amount of 3-O-sulfated pentasaccharide for the analyses by nano-electrospray and affinity coelectrophoresis. Assuming one [35S]sulfate is transferred to every molecule of pentasaccharide by 3-OST-1 enzyme, a total of 3 x 107 c.p.m. of pentasaccharide, equivalent to 2.1 nmole, were prepared.
Affinity chromatographypurified 3-O-sulfated pentasaccharide was further purified by PAMN-HPLC for nano-electrospray MS analysis. The 3-O-sulfated pentasaccharide was injected onto a PAMN column (0.46 x 25 cm; Waters) equilibrated with 350 mM KH2PO4 at a flow rate of 1 ml/min. The column was then eluted with a linear gradient of KH2PO4 from 350 mM to 1000 mM in 60 min, and the concentration of KH2PO4 remained at 1000 mM for an additional 20 min at a flow rate of 1 ml/min. The eluate was collected every minute, and 5 µl of the eluate was removed to mix with 7 ml of scintillation fluid (Ecolume, ICN) to determine [35S]radioactivity on a ß-counter (Packard Instrument). The 3-O-sulfated pentasaccharide emerged at 5455 min. The fractions containing 35S-radioactivity were pooled and dialyzed against 50 mM ammonium bicarbonate using 3,500 MWCO dialysis tubing. The dialyzed sample was resolved on BioGel P-6 (0.75 x 200 cm) eluted with 0.5 M ammonium bicarbonate to further remove potential salt contamination followed by dialyzing against 50 mM ammonium bicarbonate and water.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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