1. -Techniques in Glycobiology-
Analysis of
Proteoglycans
&
Glycosaminoglycans
-Techniques in Glycobiology-
NHLBI CardioPEG – Gerald W.Hart, September 17, 2013
Funded by NHLBI P01HL107153

2. Essentials of Glycobiology
Proteoglycans consist of a protein core and one or more covalently attached glycosaminoglycan chains
Chapter 16, Figure 2
Essentials of Glycobiology
Second Edition

3. Glycosaminoglycans consist of repeating disaccharide units
Chapter 16, Figure 3
Essentials of Glycobiology
Second Edition

4. Keratan sulfates contain a sulfated poly-N-acetyllactosamine chain
Chapter 16, Figure 4
Essentials of Glycobiology
Second Edition

5. Examples of chondroitin sulfate proteoglycans

6. Examples of keratan sulfate proteoglycans
Essentials of Glycobiology

7. Examples of heparan sulfate proteoglycans
Essentials of Glycobiology

8. Proteoglycan Analysis
Isolation and Analysis of Intact Proteoglycans
Identification of Core Protein – Sequence.
Site-Mapping of Proteoglycans & Other PTMs
Isolation & Characterization of Glycosaminoglycans
Digestions to Produce disaccharide repeat
Determining Repeat Composition
Sulfation
Non-reducing terminus
Sequencing of GAGs

9. Use of Denaturing Chaotropic Agents to Isolate Proteoglycans:

10. Isolation of Intact Proteoglycans
35SO4 + 3H-glucosamine
Proteoglycan Protocols
Edited by Renato V. Iozzo, MD

11. Typical Work Flow - Proteoglycans
Extraction of Proteoglycans – Typically 4M Urea or 6M GuHCl –Strong denaturing/chaotropic agents.
Membrane bound PGs require Detergents
Ion-Exchange -DEAE-Sephacel or similar anion exchange enrichment. – High negative charge.
Size Exclusion Chromatography – Typically Sepharose 4B
Analysis of GAG chains after release –
Protease Digestion
Beta-Elimination
GAG size fractionation – TSK4000, HPLC, Sephadex G200, Superose CL-6B

12. GPs PGs Negative Charge Allows Ready Separation from Other Glycans
Proteoglycan Protocols
Edited by Renato V. Iozzo, MD

13. Size Fractionation of Proteoglycans
Proteoglycan Protocols
Edited by Renato V. Iozzo, MD

14. Attachment of GAGs to Protein Core:

15. GAGs are Often Attached at SG Sites:

16. Chondroitin Sulfate Attachment Sites:

17. Biochemical Site Mapping of GAGs
Similar Approaches as Other O-Glycans eg.
Beta-Elimination/Michael Addition
MS/MS using ETD on PGs with GAGs Truncated

18. GAG Structure – 3 Regions: Linkage, Repeat, Non-Reducing Terminus:

19. Analysis of Glycosaminoglycans
Release from PGs – Protease, Beta-Elimination
Lyases & Hydrolases – Fragment GAGs
Disaccharide Compostion Analysis
Sulfation Sites
Non-Reducing Terminus
Mercuric acetate elimination of unsaturated bond containing disaccharides reveals non-reducing Terminus
Presence of Classical N- & O-Glycans
Linkage of oligosaccharides
O-Glycans in beta-eliminated GAGS

20. GAG Degrading Enzymes: Hydrolases & Eliminases:
Hydrolase – Catalyzes Hydrolysis i.e. Addition of water across a chemical bond.
A-B + H2O  A-OH + B-H
Examples: testicular Hyase; endo-b-galactosidase
Eliminase – Catalyzes the removal of H2O from a chemical bond.
A-B  A-OH + dB + H2O
Examples: chondroitinase ABC, HS lyases, Strept. Hyaluronidase.

21. Characterization of Glycosaminoglycans:
Bacterial Eliminases
Are
Powerful Tools:
Sequential
Degradation
Followed by
Gel Filtration.
2. Other Separation
Methods.

22. Degrades All Chondroitin Sulfates, Dermatan Sulfates and Hyaluronic Acid

23. Digests All Types of Chondroitin Sulfates and Hyaluronic Acids, but Not Digest Hyaluronic Acids.

25. Hyaluronidases (eg. testicular; a hydrolyase)
Also Degrades
Chondroitin sulfates

28. Keratanases
Essentials of Glycobiology

29. Heparinase Specificities

30. Heparin Fragments on a 20% Acrylamide Gel:

31. Typical Repeating Disaccharides

32. Nitrous Acid Degradation of Heparan Sulfate & Heparin
Nat. Prod. Rep. , 2002, 19,

33. Analytical Methods for GAG Repeat Disaccharides
Paper Chromatography
Thin Layer Chromatography
HPLC Methods
Capillary Electrophoresis
Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)

34. Disaccharides Released by Chondroitinase:

35. Paper Chromatography of Released Disaccharides

36. Thin-Layer Chromatography of Released Disaccharides
Silica Gel 60 TLC aluminum plate and developed with a solvent
system consisting of n -butanol/formic acid/water (4:8:1,).

37. Attaching a Chromophore for Analysis:
Sigma Chemical Company

38. HPLC separation of CS-derived saturated and unsaturated
disaccharides labeled with 2AB
FIG. 2. HPLC separation of CS-derived saturated and unsaturated
disaccharides labeled with 2AB. CS-derived saturated tetrasaccharides
Tetra-C1–C4 were individually digested with chondroitinase
AC-II and derivatized with 2AB. The four digests and 2ABderivatized
unsaturated CS-disaccharides were mixed and analyzed
by HPLC as described in the legend to Fig. 1. The HPLC conditions
were the same as those for the experiments shown in Fig. 1.

39. Separation of AMAC-Labeled Disaccharides by RP-HPLC:HS CS

40. Disaccharides from Rat Liver GAGs-SAX-HPLC
Heparinases
CS-ABCase

41. GAG Disaccharides from MDCK Cells
Heparinase
CSase ABC

42. 2-aminobenzamide (2AB) labeled Disaccharides on Anion Exchange Columns:
STDs
Brain
Cartilage
Skin

43. Anion-Exchange Analysis of Linkage Region:

44. Scheme for Sequencing CS:

45. FACE Analysis of Disaccharides

46. FACE Analysis of GAG-Derived Disaccharides:

47. Identifying the Non-Reducing Ends

48. Using Mercuric Acetate to ID Reducing Ends:

49. Using FACE to Analyze Non-Reducing Ends:

50. Specific Enzymes to Confirm Sulfation:

51. Specific Enzymes to Confirm Sulfation:

52. Biosynthesis of Chondroitin Sulfate
NATURE CHEMICAL BIOLOGY | VOL 7 | NOVEMBER 2011

53. Difficulties in Sequencing GAGs:
Lack of sufficient quantities of pure proteoglycans,
the multiple sequences possible for the multiple GAG chains often present on a single core protein,
the difficulties in purifying a single GAG chain for sequencing
difficulties in determining GAG sequence.
Why Bikunin:
Bikunin is a member of the kunin family of serine protease inhibitors13–15, is a therapeutically relevant proteoglycan that is used in Japan as a drug for the treatment of acute pancreatitis. Thus, bikunin is available at a high level of purity in multimilligram quantities.
Bikunin has the simplest chemical structure of any proteoglycan, with a single site for O-linked modification by a GAG chain, located at Ser10 in its 16-kDa core protein.
The protein component of bikunin is well characterized, but its GAG chain structure is heterogeneous and has received less attention because of the technical difficulties associated with GAG analysis.
GAG chain is quite short, it is very heterogeneous in size and composition, with 27–39 saccharide residues and a molecular mass (MR) ranging from 5,505 Da to 7,102 Da23.
In addition, enzymatic analysis shows that the bikunin GAG chains contain single-uronic-acid stereochemistry (glucuronic acid), sulfo groups at only the 4 position of its galactosamine residue and no N-sulfo group or N-acetyl group variability, which is common in the GAG chains of the more structurally complex members of the heparan sulfate proteoglycan family.

54. MS/MS FT-ICR-MS CS GAG Complete conversion to Na salt
NATURE CHEMICAL BIOLOGY | VOL 7 | NOVEMBER 2011

55. FT-ICR negative-ion mass spectrum of 5
FT-ICR negative-ion mass spectrum of 5.80-kDa MR fractionby PAGE with 18 isobars and 63 parent ions.
Deconvolution
CID-FT-ICR-MS/MS spectra of parent-ion m/z =
fraction. (a) FT-ICR negative-ion mass spectrum of 5.80-kDa MR fraction
by PAGE with 18 isobars and 63 parent ions. (b) Deconvolution of
spectrum a. (c) CID-FT-ICR-MS/MS spectra of parent-ion m/z =
(z = 6) and annotated fragment-ions providing sequence with dp27-5-Ser
fragmentation pattern assigned from spectrum.
Sequencing by CID
NATURE CHEMICAL BIOLOGY | VOL 7 | NOVEMBER 2011

56. Flow Chart for Analysis:
Flow chart reads from left to right. The MR (kDa) determined based on PAGE of fractions (blue rectangles represent gel bands) of bikunin in peptidoglycosaminoglycan prepared by continuous elution PAGE is shown. The deconvoluted MS obtained using FT-ICR-MS affords the mass of 3–5 odd and even components (green ovals) observed in each bikunin peptidoglycosaminoglycan fraction is shown. Each MS spectrum showed multiple charge states (z values) shown as red diamonds from which parent ions were selected for MS/MS giving CID fragments by analysis on FT (purple circles) or FT-ICR (brown circles). A shorthand sequence for each chain is shown with a, b, c and d subdomain repeats indicated by numbers (that is, for d = 0, c = 2, b = 0, a = 3). The overall sequence of bikunin chondroitin sulfate-A peptidoglycosaminoglycan shown at the bottom is consistent with all determined sequences.
The proteoglycan bikunin has a defined sequence
Mellisa Ly, Franklin E Leach III, Tatiana N Laremore, Toshihiko Toida, I Jonathan Amster & Robert J Linhardt
Nature Chemical Biology 7, 827–833 (2011) doi: /nchembio.673

57. Analysis of Proteoglycans
Advances in Molecular Biology Have Allowed a Detailed Understanding of Core Proteins.
Site Mapping is Similar to other O-Glycans.
GAG Analysis is Greatly Facilitated by the High Specificity of Bacterial Lyases.
Detailed Sequencing of GAGs is still Very Difficult.
Current Technology is NOT Capable of Defining the molecular Species of a Proteoglycan = Information Content.
Recent Developments in Mass Spectrometry are Showing Promise.