Electrophoresis

1. Immediately before electrophoresis, rinse the resolving gel surface with stacking gel buffer (0.125 M Tris-HC1 buffer, pH 6.8, diluted from the 1 M stock solution).

2. Prepare and degas the stacking gel solution (for 5 ml, mix 0.5 mL of T50%/C5% acrylamide stock with 0.6 mL of 1 M Tris pH 6.8 and 3.9 mL of distilled water).

Proteoglycan Gel

Fig. 1. Principles of integral glycan sequencing and an example. (A) Fluorescence detection of different amounts of a 2AA-tagged heparin tetrasaccharide run on a 33% minigel. (B) Exosequencing of a 2AA-tagged heparin tetrasaccharide with lysosomal enzymes and separation of the products on a 33% minigel (15 pmol per track). Band shifts following the exoenzyme treatments shown reveal the structure of the nonreducing end disaccharide unit (track 1, untreated). I2Sase, iduronate-2-sulfatase; Idase, iduronidase; G6Sase, glucosamine-6-sulfa-tase; Nsase, sulfamidase. (C) Schematic representation of IGS of a hexasaccharide (pHNO2, partial nitrous acid treatment). (D) Actual example of IGS performed on a purified heparin hexasaccharide, corresponding to the scheme in (C), using the combinations of pHNO2 and exoenzyme treatments indicated (track 1, untreated, 25 pmol; other tracks correspond to ~200 pmol/per track of starting sample for pHNO2 digest). The hexasaccharide (purified from bovine lung heparin) has the putative structure IdoA(2S)-GlcNSO3(6S)-IdoA(2S)-GlcNSO3(6S)-IdoA(2S)-AMannR(6S). Electrophoresis was performed on a 16 cm 35% gel. Copyright © 1999 National Academy of Sciences, USA. From ref. (11).

Fig. 1. Principles of integral glycan sequencing and an example. (A) Fluorescence detection of different amounts of a 2AA-tagged heparin tetrasaccharide run on a 33% minigel. (B) Exosequencing of a 2AA-tagged heparin tetrasaccharide with lysosomal enzymes and separation of the products on a 33% minigel (15 pmol per track). Band shifts following the exoenzyme treatments shown reveal the structure of the nonreducing end disaccharide unit (track 1, untreated). I2Sase, iduronate-2-sulfatase; Idase, iduronidase; G6Sase, glucosamine-6-sulfa-tase; Nsase, sulfamidase. (C) Schematic representation of IGS of a hexasaccharide (pHNO2, partial nitrous acid treatment). (D) Actual example of IGS performed on a purified heparin hexasaccharide, corresponding to the scheme in (C), using the combinations of pHNO2 and exoenzyme treatments indicated (track 1, untreated, 25 pmol; other tracks correspond to ~200 pmol/per track of starting sample for pHNO2 digest). The hexasaccharide (purified from bovine lung heparin) has the putative structure IdoA(2S)-GlcNSO3(6S)-IdoA(2S)-GlcNSO3(6S)-IdoA(2S)-AMannR(6S). Electrophoresis was performed on a 16 cm 35% gel. Copyright © 1999 National Academy of Sciences, USA. From ref. (11).

3. Add 10% ammonium persulfate (10 ^L) and TEMED (5 ^L). Immediately pour on to the top of the resolving gel and insert the well-forming comb.

4. After polymerization (~15 min), remove the comb and rinse the wells thoroughly with electrophoresis buffer.

5. Place the gel unit into the electrophoresis tank and fill the buffer chambers with electro-phoresis buffer.

6. Load the oligosaccharide samples (5-20 ^L depending on well capacity, containing ~10% (v/v) glycerol or sucrose in 125 mM Tris-HCl, pH 6.8, carefully into the wells with a microsyringe. Marker samples containing bromophenol blue and phenol red should also be loaded into separate tracks.

7. Run the samples into the stacking gel at 150-200 V (typically, 20-30 mA) for 30-60 min, followed by electrophoresis at 300-400 V (typically 20-30 mA and decreasing during run) for approximately 5-8 h (for a 16 cm gel). Heat generated during the run should be dissipated using a heat exchanger with circulating tap water, or by running the gel in a cold room or in a refrigerator.

8. Electrophoresis should be terminated before the Phenol red marker dye is about 5 cm from the bottom of the gel. (At this point, disaccharides should be 3-4 cm from the bottom of the gel.)

Phenol Gel Electrophoresis

Fig. 2. IGS of a heparin hexasaccharide of known structure. A heparin hexasaccharide with the structure DHexA(2S)-GlcNSO3(6S)-IdoA-GlcNAc (6S)-GlcA-GlcNSO3(6S), was 2AA-tagged and subjected to sequencing on 16cm 33% gel. (A) IGS of hexasaccharide using the combinations of pHNO2 and exoenzyme treatments indicated (track 1, untreated, 20 pmol; other tracks correspond to ~90 pmol/per track of starting sample for pHNO2 digest). NAG, N-acetylglucosaminidase. (B) Determining the sequence of the nonreducing disaccharide unit of the hexasaccharide using the I2Sase, G6Sase, and mercuric acetate (MA) treatments shown (~20 pmol/track; track 1, untreated). Copyright © 1999 National Academy of Sciences, USA. From ref. (11).

Fig. 2. IGS of a heparin hexasaccharide of known structure. A heparin hexasaccharide with the structure DHexA(2S)-GlcNSO3(6S)-IdoA-GlcNAc (6S)-GlcA-GlcNSO3(6S), was 2AA-tagged and subjected to sequencing on 16cm 33% gel. (A) IGS of hexasaccharide using the combinations of pHNO2 and exoenzyme treatments indicated (track 1, untreated, 20 pmol; other tracks correspond to ~90 pmol/per track of starting sample for pHNO2 digest). NAG, N-acetylglucosaminidase. (B) Determining the sequence of the nonreducing disaccharide unit of the hexasaccharide using the I2Sase, G6Sase, and mercuric acetate (MA) treatments shown (~20 pmol/track; track 1, untreated). Copyright © 1999 National Academy of Sciences, USA. From ref. (11).

3.5. Imaging the Gels

Effective gel imaging requires a CCD camera that can detect faint fluorescent banding patterns by capturing multiple frames. Systems commonly used for detection of ethidium bromide stained DNA can usually be adapted with appropriate filters as described in Note 6.

1. Place a UV filter (UG-1, UG-11, or MUG-2) onto the transilluminator, and fit a 450-nm blue filter onto the camera lens.

2. Remove the gel carefully from the glass plates after completion of the run and place on the UV transilluminator surface wetted with electrophoresis buffer. Also wet the upper surface of the gel to prevent gel drying and curling.

3. Switch on transilluminator and capture image using CCD camera. Exposure times are typically 1-5 s depending on the amount of labeled saccharide (see Note 7).

3.6. Interpreting the Data

The sequence of saccharides subjected to IGS can be read directly from the banding pattern by interpreting the band shifts due to removal of specific sulfate or sugar

Hplc Saccharides Peak

Fig. 3. Purification and IGS of a HS decasaccharide. (A) SAX-HPLC of a pool of HS decasaccharides derived by heparitinase treatment of porcine mucosal HS. For details of this technique, see Chapter 14. The arrowed peak was selected for sequencing. (B) IGS of the purified HS decasaccharide on a 16 cm 33% gel using the combinations of pHNO2 and exoenzyme treatments indicated (track 1, untreated, 20 pmol; other tracks correspond to ~400 pmol/per track of starting sample for pHNO2 digest). (C) Determining the sequence of the nonreducing disac-charide unit of the HS decasaccharide using the mercuric acetate (MA) and G6Sase treatments shown (approximately 40 pmol per track; track 1, untreated). Copyright© 1999 National Academy of Sciences, USA. From ref. (11).

Fig. 3. Purification and IGS of a HS decasaccharide. (A) SAX-HPLC of a pool of HS decasaccharides derived by heparitinase treatment of porcine mucosal HS. For details of this technique, see Chapter 14. The arrowed peak was selected for sequencing. (B) IGS of the purified HS decasaccharide on a 16 cm 33% gel using the combinations of pHNO2 and exoenzyme treatments indicated (track 1, untreated, 20 pmol; other tracks correspond to ~400 pmol/per track of starting sample for pHNO2 digest). (C) Determining the sequence of the nonreducing disac-charide unit of the HS decasaccharide using the mercuric acetate (MA) and G6Sase treatments shown (approximately 40 pmol per track; track 1, untreated). Copyright© 1999 National Academy of Sciences, USA. From ref. (11).

moieties. Figure 1 shows an actual example and a schematic representation. First, bands generated by the partial nitrous acid treatment indicate the positions of N-sul-fated glucosamine residues in the original saccharide (see Fig. 1C, track 2). Lack of a band at a particular position indicates the presence of an N-acetylated glucosamine residue (an example of this is shown in Fig. 2). Such saccharides can be sequenced with the additional use of the exoenzyme N-acetylglucosaminidase, which removes this residue and allows further sequencing of an otherwise "blocked" fragment. Following the nitrous acid treatment, the "ladder" of bands is then subjected to various exoenzyme digestions. The presence of specific sulfate or sugar residues can be deduced from the band shifts that occur (see Fig. 1C, tracks 3-5). Figure 3 shows an example of a decasaccharide from HS which has been purified by SAX-HPLC and sequenced using IGS.

Usually the band shifts are downwards, due to the lower molecular mass and thus higher mobility of the product. However, it should be noted that occasionally upward shifts occur, probably due to subtle differences in charge/mass ratio (for examples, see Figs. 1B, 2B and 3C). Note also that minor "ghost" bands sometimes appear after the nitrous acid treatment. They are probably due to loss of an N-sulfate group, and normally these do not affect interpretation of the shifts in the major bands (11).

If the saccharide being sequenced was derived by bacterial lyase treatment, it will have a A-4,5-unsaturated uronate residue at its nonreducing terminus. If this residue has a 2-O-sulfate attached, this can be detected by susceptibility to I2Sase (see Fig. 2B), but the sugar residue itself is resistant to both Idase and Gase. Its removal is required in order to confirm whether there is a 6-O-sulfate on the adjacent non-reducing end glucosamine (see Figs. 2B and 3C for examples). However, bacterial enzymes which specifically remove the A-4,5-unsaturated uronate residues (and the 2-O-sulfate groups that may be present on them) are now available commercially (see Table 1). Alternatively, they can be removed chemically with mercuric acetate (20; see Figs. 2B and 3C).

In addition to the basic sequencing experiment, it is wise to confirm agreement of the data with an independent analysis of the disaccharide composition of the saccha-ride (see Chapter 14). It can sometimes be difficult to sequence the reducing terminal monosaccharide, due to it being a poor substrate for the exoenzymes. In these cases it has proved more effective to analyze the terminal 2AA-labeled disaccharide unit in comparison to 2AA-labeled disaccharide standards (11).

4. Notes

1. Using large excesses of reagent as described, saccharides derived from HS and heparin by bacterial lyase scission generally couple with 2-AA with efficiencies in the range 60-70%. In contrast, saccharides derived from HS and heparin by low-pH nitrous acid scissioning (i.e., having an anhydromannose residue at their reducing ends) label more efficiently (~70-80% coupling efficiency).

2. Unwanted reactants and solvent can also be removed from labeled saccharides by methods such as dialysis, but the rapid gel filtration chromatography step described above using the HiTrap desalting columns is convenient and usually allows good recoveries of loaded sample, particularly for 2-AA-labeled saccharides (~80%).

3. It is useful to perform some trial incubations to test for optimal time points needed to generate a balance of all fragments in the partial nitrous acid digestion. With longer saccharides (octasaccharides and larger) it is observed that the largest products are generated quickly and thus a bias toward shorter incubations is required as saccharide length increases.

4. The enzyme conditions should provide for complete digestion of all susceptible residues. This is important to the sequencing process, since incomplete digestion would create a more complex banding pattern and would give a false indication of sequence heterogeneity. It is useful to run parallel controls with standard saccharides to enable monitoring of reaction conditions. When combinations of exoenzymes are required, these can be incubated simultaneously with the sample. If necessary, the activity of one enzyme can be destroyed before a secondary digestion with a different enzyme by heating the sample at 100°C for 2-5 min.

5. Adequate separations, particularly over limited size ranges of saccharides, can be obtained using single concentration gels, typically in the range 25-35% acrylamide. Improvements in resolution can be made by using longer gel sizes. Different voltage conditions (usually in the range 200-600 V) and running times are required for different gel formats, and should be established by trial and error with the particular samples being analyzed. Gels up to 24 cm in length can usually be run in 5-8 h using high voltages, whereas with longer gels it is more convenient to use lower voltage conditions and run overnight. We have also found that minigels can also be used effectively for separation of HS/heparin saccharides (see Fig. 1). Note that it is also possible to run Tris-acetate gels with a Tris-MES electrophoresis buffer (see Fig. 1; 11).

6. Because the emission wavelength is 410-420 nm, there is a need to filter out background visible wavelength light from the UV lamps. This can be done effectively with special glass filters that permit transmission of UV light but do not allow light of wavelengths >400 nm to pass. A blue bandpass filter on the camera also improves sensitivity. Suitable filters are available from HV Skan (Stratford Road, Solihull, UK; Tel: 0121 733 3003) or UVItec Ltd (St Johns Innovation Centre, Cowley Road, Cambridge, UK: www.uvitec. demon.co.uk).

7. Required exposure times are strongly dependent on sample loading and the level of detection required. Overly long exposures will result in excessive background signal. Note that negative images are usually better for band identification (see figures). Under the conditions described, the limit of sensitivity is ~1-2 pmol/band (see Fig. 1), with ~5-10 pmol being optimal. Recently it has been found that ~10-fold more sensitive detection is possible using an alternative fluorophore aminonaphthalenedisulfonic acid (21,22).

References

1. Spillmann, D. and Lindahl, U. (1994) Gycosaminoglycan-protein interactions: a question of specificity. Curr. Opin. Cell Biol. 4, 677-682.

2. Bernfield M., Gotte, M., Park, P. W., et al. Functions of Cell Surface Heparan Sulfate Proteoglycans. Ann. Rev. Biochem. (1999) 68, 729-777.

3. Turnbull, J. E. and Gallagher, J. T. (1991) Distribution of Iduronate-2-sulfate residues in HS: evidence for an ordered polymeric structure. Biochem. J. 273, 553-559.

4. Turnbull, J. E., Fernig, D., Ke, Y., Wilkinson, M. C., and Gallagher, J. T. (1992) Identification of the basic FGF binding sequence in fibroblast HS. J. Biol. Chem. 267, 10,337-10,341.

5. Pervin, A., Gallo, C., Jandik, K., Han, X., and Linhardt, R., (1995) Preparation and structural characterisation of heparin-derived oligosaccharides. Glycobiology 5, 83-95.

6. Yamada, S., Yamane, Y., Tsude, H., Yoshida, K., and Sugahara, K. (1998) A major common trisulfated hexasaccharide isolated from the low sulfated irregular region of porcine intestinal heparin. J. Biol. Chem, 273, 1863-1871.

7. Yamada, S., Yoshida, K., Sugiura, M., Sugahara, K., Khoo, K., Morris, H., and Dell, A. (1993) Structural studies on the bacterial lyase-resistant tetrasaccharides derived from the antithrom-bin binding site of porcine mucosal intestinal heparin. J. Biol. Chem. 268, 4780-4787.

8. Mallis, L., Wang, H., Loganathan, D., and Linhardt, R. (1989) Sequence analysis of highly sulfated heparin-derived oligosaccharides using FAB-MS. Anal. Chem. 61, 1453-1458.

9. Rhomberg A. J., Ernst, S., Sasisekharan, R., Biemann, K., et al. (1998) Mass spectromet-ric and capillary electrophoretic investigation of the enzymatic degradation of heparin-like glycosaminoglycans. Proc. Natl. Acad. Sci. (USA) 95, 4176-4181.

10. Hopwood, J. (1989) Enzymes that degrade heparin and heparan sulfate. In: Heparin (Lane and Lindahl, eds.), Edward Arnold, London, UK, pp. 191-227.

11. Turnbull, J. E., Hopwood, J. J., and Gallagher, J. T. (1999) A strategy for rapid sequencing of heparan sulfate/heparin saccharides. Proc. Natl. Acad. Sci. (USA) 96, 2698-2703.

12. Merry, C. L. R., Lyon, M., Deakin, J. A., Hopwood, J. J., and Gallagher, J. T. (1999) Highly sensitive sequencing of the sulfated domains of heparan sulfate. J. Biol. Chem. 274, 18,455-18,462.

13. Vives, R. R., Pye, D. A., Samivirta, M., Hopwood, J. J., Lindahl, U., and Gallagher, J. T. (1999) Sequence analysis of heparan sulphate and heparin oligosaccharides. Biochem. J. 339, 767-773.

14. Venkataraman, G., Shriver, Z., Raman, R., Sasisekharan, R., et al. (1999) Sequencing complex polysaccharides. Science 286, 537-542.

15. Guimond, S. E. and Turnbull, J. E. (1999) Fibroblast growth factor receptor signalling is dictated by specific heparan sulfate saccharides. Curr. Biol. 9, 1343-1346.

16. Shively, J. and Conrad, H. (1976) Formation of anhydrosugars in the chemical depolymerisation of heparin. Biochemistry 15, 3932-3942.

17. Bienkowski, M. J. and Conrad, H. E. (1985) Structural characterisation of the oligosaccharides formed by depolymerisation of heparin with nitrous acid. J. Biol. Chem. 260, 356-365.

18. Turnbull, J. E. and Gallagher, J. T. (1988) Oligosaccharide mapping of heparan sulfate by polyacrylamide-gradient-gel electrophoresis and electrotransfer to nylon membrane. Biochem J 251, 597-608.

19. Rice, K., Rottink, M., and Linhardt, R. (1987) Fractionation of heparin-derived oligosaccharides by gradient PAGE. Biochem. J. 244, 515-522.

20. Ludwigs, U., Elgavish, A., Esko, J., and Roden, L. (1987) Reaction of unsaturated uronic acid residues with mercuric salts. Biochem. J. 245, 795-804.

21. Drummond, K. J., Yates, E. A., and Turnbull. J. E. (2001) Electrophoretic sequencing of heparin/heparin sulfate oligosaccharides using a highly sensitive fluorescent end label. Proteomics (in press).

22. Lee, K. B., Al-Hakim, A., Loganathan, D., and Linhardt, R. J. (1991) A new method for sequencing linear oligosaccharides on gels using charged fluorescent conjugates. Carbohydr.Res. (1991) 214, 155-168

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