Sequencing complex polysaccharides

Science

Volumn 286, Issue 5439, 1999, Pages 537-542

  Venkataraman, Ganesh ^a   Shriver, Zachary ^b   Raman, Rahul ^b   Sasisekharan, Ram ^b  

^a HARVARD MIT DIVISION OF HEALTH SCIENCES AND TECHNOLOGY   (United States)

^b MASSACHUSETTS INSTITUTE OF TECHNOLOGY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

GLYCOSAMINOGLYCAN;

ARTICLE; CARBOHYDRATE ANALYSIS; MASS SPECTROMETRY; MOLECULAR WEIGHT; PRIORITY JOURNAL;

CARBOHYDRATE SEQUENCE; DISACCHARIDES; GLYCOSAMINOGLYCANS; HEPARIN; HEPARIN LYASE; MOLECULAR SEQUENCE DATA; MOLECULAR WEIGHT; NITROUS ACID; OLIGOSACCHARIDES; POLYSACCHARIDE-LYASES; SEQUENCE ANALYSIS; SPECTROMETRY, MASS, MATRIX-ASSISTED LASER DESORPTION-IONIZATION;

EID: 0033569397     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5439.537     Document Type: Article

References (39)

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23. The hexadecimal system chosen here and shown in Fig. 1B is based on a property-encoded notation system (R. Raman et al., in preparation).
24. Here, the disaccharide building blocks of HLGAGs are first represented with the uronic acid at the nonreducing end and the hexosamine at the reducing end (U-H). It is also possible to have H at the nonreducing end to define the repeating disaccharides in the HLGAG chain (H-U), although this will not conceptually change the analysis.
25. The methodology used for the analysis of acidic polysaccharides involves the detection of these saccharides as noncovalent complexes with a basic peptide or protein. Interactions between the polysaccharide and the protein or peptide allow for the ionization of normally labile HLGAGs as intact species and their detection in the positive ion mode. With the mass spectrometer in the linear mode, this technique is able to detect as little as 100 fmol of material [P. Juhasz and K. Biemann, Carbohydr. Res. 270, 131 (1995); P. Juhasz and K. Biemann, Proc. Natl. Acad. Sci. U.S.A. 91, 4333 (1994); A. J. Rhomberg et al., ibid. 95, 4176 (1998); S. Ernst et al., ibid., p. 4182; A. J. Rhomberg et al., ibid., p. 12232].
26. The methodology used for the analysis of acidic polysaccharides involves the detection of these saccharides as noncovalent complexes with a basic peptide or protein. Interactions between the polysaccharide and the protein or peptide allow for the ionization of normally labile HLGAGs as intact species and their detection in the positive ion mode. With the mass spectrometer in the linear mode, this technique is able to detect as little as 100 fmol of material [P. Juhasz and K. Biemann, Carbohydr. Res. 270, 131 (1995); P. Juhasz and K. Biemann, Proc. Natl. Acad. Sci. U.S.A. 91, 4333 (1994); A. J. Rhomberg et al., ibid. 95, 4176 (1998); S. Ernst et al., ibid., p. 4182; A. J. Rhomberg et al., ibid., p. 12232].
27. The methodology used for the analysis of acidic polysaccharides involves the detection of these saccharides as noncovalent complexes with a basic peptide or protein. Interactions between the polysaccharide and the protein or peptide allow for the ionization of normally labile HLGAGs as intact species and their detection in the positive ion mode. With the mass spectrometer in the linear mode, this technique is able to detect as little as 100 fmol of material [P. Juhasz and K. Biemann, Carbohydr. Res. 270, 131 (1995); P. Juhasz and K. Biemann, Proc. Natl. Acad. Sci. U.S.A. 91, 4333 (1994); A. J. Rhomberg et al., ibid. 95, 4176 (1998); S. Ernst et al., ibid., p. 4182; A. J. Rhomberg et al., ibid., p. 12232].
28. The methodology used for the analysis of acidic polysaccharides involves the detection of these saccharides as noncovalent complexes with a basic peptide or protein. Interactions between the polysaccharide and the protein or peptide allow for the ionization of normally labile HLGAGs as intact species and their detection in the positive ion mode. With the mass spectrometer in the linear mode, this technique is able to detect as little as 100 fmol of material [P. Juhasz and K. Biemann, Carbohydr. Res. 270, 131 (1995); P. Juhasz and K. Biemann, Proc. Natl. Acad. Sci. U.S.A. 91, 4333 (1994); A. J. Rhomberg et al., ibid. 95, 4176 (1998); S. Ernst et al., ibid., p. 4182; A. J. Rhomberg et al., ibid., p. 12232].
29. The methodology used for the analysis of acidic polysaccharides involves the detection of these saccharides as noncovalent complexes with a basic peptide or protein. Interactions between the polysaccharide and the protein or peptide allow for the ionization of normally labile HLGAGs as intact species and their detection in the positive ion mode. With the mass spectrometer in the linear mode, this technique is able to detect as little as 100 fmol of material [P. Juhasz and K. Biemann, Carbohydr. Res. 270, 131 (1995); P. Juhasz and K. Biemann, Proc. Natl. Acad. Sci. U.S.A. 91, 4333 (1994); A. J. Rhomberg et al., ibid. 95, 4176 (1998); S. Ernst et al., ibid., p. 4182; A. J. Rhomberg et al., ibid., p. 12232].
30. 2s in highly sulfated regions of HLGAGs, whereas heparinase III can clip at I in unsulfated regions of HLGAGs. Using defined substrates, we defined the cleavage conditions for heparinase I and III under our reaction conditions. We found that there were certain well-defined parameters under which heparinase I and III clip at their respective primary sites and not at their secondary sites (supplementary data is available at Science Online at www. sciencemag.org/feature/data/1042118.shl). In part, these studies defined the designated "short" and "exhaustive" reaction conditions outlined below ( 18). In each case, through the use of our sequencing strategy and independent experimental constraints (that is, incomplete nitrous acid followed by exoenrymes or exhaustive nitrous acid and compositional analysis), we can confirm the sequence assignment obtained with heparinases as experimental constraints.
31. Other methods including the use of exoenzymes are being developed for sequencing saccharides, and for the most part require sample isolation and repurification [J. H. Turnbull et al., Proc. Natl. Acad. Sci. U.S.A. 96, 2703 (1999), and references therein; R. Vives et al., Biochem. J. 339, 767 (1999)]. However, the MALDI-MS approach for sequencing saccharides with exoenzymes does not require repurification of the saccharide after manipulations, The MALDI mass spectrogram is able to generate all the sequence information of all the saccharide fragments in a "ladder" form in one spectrum.
32. Other methods including the use of exoenzymes are being developed for sequencing saccharides, and for the most part require sample isolation and repurification [J. H. Turnbull et al., Proc. Natl. Acad. Sci. U.S.A. 96, 2703 (1999), and references therein; R. Vives et al., Biochem. J. 339, 767 (1999)]. However, the MALDI-MS approach for sequencing saccharides with exoenzymes does not require repurification of the saccharide after manipulations, The MALDI mass spectrogram is able to generate all the sequence information of all the saccharide fragments in a "ladder" form in one spectrum.
33. The AT-III binding decasaccharide was a gift from R. Linhardt. The sequence of this decasaccharide has been reported to be D4-7DD, on the basis of NMR spectroscopy [Y. Toida et al., J. Biol. Chem. 271, 32040 (1996)]. Such a sequence should show the appearance of a tagged D or DD residue at the reducing end. However, we find all the different experiments used in the elucidation of the decasaccharide sequence to be consistent with each other in the appearance of a 4-7 tagged product and not a D (or a DD) product. This saccharide does not contain an intact AT-III binding site, as proposed. Therefore, we sought confirmation of our proposed sequence through the use of integral glycan sequencing (IGS), which agreed with our analysis.
34. NAc is a N-acetytated galactosamine, and there are 16 disaccharide building blocks for DCMP. Like the heparinases that degrade HLGAGs, there are distinct chondoroitinases and other chemical methods available that clip at specific glycosidic linkages of DCMP and serve as experimental constraints. Furthermore, because DCMPs are acidic polysaccharides, the MALDI-MS techniques and methods used for HLGAGs can be readily extended to the DCMPs.
35. The monomeric units of NAN and NCN are linked by α 2-8 glycosidic linkages and can be modified at the 4-O, 7-O, and 9-O positions. Methods of purifying and characterizing PSA oligosaccharides using high-performance liquid chromatography, CE, and MS have very recently been established. In addition, chemical and exosialydases and endosialydases that cleave at distinct glycosidic linkage of PSA are available and can serve as experimental constraints.
36. The sequencing approach presently uses a brute force method because many of the rules regarding the specificity of enzymes that degrade and modify complex polysaccharides are in the developmental stage. Once these rules or constraints are fully developed, more intelligent algorithms such as a genetic algorithm or Monte Carlo optimization could be used to search a much narrower search space for the correct sequence. The combination of more efficient constraints and algorithms will thus lead to a fully automated sequencing approach.
37. The use of heparinases was essentially as described in references in (77). Digests were designated as either short or exhaustive. Short digests were completed with 50 nM enzyme for 10 min. Exhaustive digests were completed with 200 nM enzyme for either 4 hours or overnight. Partial nitrous acid cleavage was completed using a modification of published procedures. Briefly, to an aqueous solution of saccharide was added a 2X solution of sodium nitrite in HCl so that the concentration of nitrous acid was 2 mM and that of HCl was 20 mM. The reaction was allowed to proceed at room temperature with quenching of aliquots at various time points through the addition of 1 μl of 200 mM sodium acetate and of bovine serum albumin (1 mg/ml; pH 6.0). Exhaustive nitrous acid cleavage was completed by reacting saccharide with 4 mM nitrous acid in HCl overnight at room temperature. In both cases, it was found that the products of nitrous acid cleavage could be sampled directly by MALDI without further cleanup and without the need to reduce the anhydromannose residues to anhydromannitol. The entire panel of HLGAG-degrading exoenzymes was purchased from Oxford Glycosystems (Wakefield, MA) and used as suggested by the manufacturer.
38. G. Venkataraman et al., Proc. Natl. Acad. Sci. U.S.A. 96, 1892 (1999).
39. Supported in part by the Massachusetts Institute of Technology and NIH (grant number GM 57073). We thank K. Biemann for help and input on the MALDI-MS; K. Pojasek for technical help with MS; R. Linhardt and J. Turnbull for saccharides and corroboration of our AT-III decasaccharide microsequencing results with exoenzyme sequencing (IGS); and J. Essigmann, A. Grodzinsky, R. Langer, and V. Sasisekharan for critical comments.