-Techniques in Glycobiology-NHLBI CardioPEG – Gerald W.Hart, September 17, 2013

Funded by NHLBI P01HL107153

Analysis ofProteoglycans

&Glycosaminoglyc

ans

2

Proteoglycans consist of a protein core and one or more covalently attached glycosaminoglycan chains

Chapter 16, Figure 2Essentials of Glycobiology

Second Edition

3

Glycosaminoglycans consist of repeating disaccharide units

Chapter 16, Figure 3Essentials of Glycobiology

Second Edition

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Keratan sulfates contain a sulfated poly-N-acetyllactosamine chain

Chapter 16, Figure 4Essentials of Glycobiology

Second Edition

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Examples of chondroitin sulfate proteoglycans

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Examples of keratan sulfate proteoglycans

Essentials of Glycobiology

Examples of heparan sulfate proteoglycans

7Essentials of Glycobiology

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 8

Use of Denaturing Chaotropic Agents to Isolate Proteoglycans:

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Isolation of Intact Proteoglycans

10Proteoglycan ProtocolsEdited by Renato V. Iozzo, MD

35SO4 + 3H-glucosamine

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 DigestionBeta-Elimination

GAG size fractionation – TSK4000, HPLC, Sephadex G200, Superose CL-6B

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12Proteoglycan ProtocolsEdited by Renato V. Iozzo, MD

Negative Charge Allows Ready Separation from Other Glycans

PGsGPs

Size Fractionation of Proteoglycans

13Proteoglycan ProtocolsEdited by Renato V. Iozzo, MD

Attachment of GAGs to Protein Core:

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GAGs are Often Attached at SG Sites:

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Chondroitin Sulfate Attachment Sites:

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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

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

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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-GlycansLinkage of oligosaccharidesO-Glycans in beta-eliminated GAGS

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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.

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Characterization of Glycosaminoglycans:

Bacterial EliminasesAre Powerful Tools:

1. Sequential Degradation Followed by Gel Filtration.

2. Other Separation Methods.

22Degrades All Chondroitin Sulfates, Dermatan Sulfates and

Hyaluronic Acid

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

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Hyaluronidases (eg. testicular; a hydrolyase)

Also DegradesChondroitin sulfates

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Keratanases

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Essentials of Glycobiology

Heparinase Specificities

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Heparin Fragments on a 20%

Acrylamide Gel:

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Typical Repeating Disaccharides

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Nitrous Acid Degradation of Heparan Sulfate & Heparin

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Nat. Prod. Rep. , 2002, 19, 312-331

Analytical Methods for GAGRepeat Disaccharides

Paper ChromatographyThin Layer ChromatographyHPLC MethodsCapillary ElectrophoresisFluorophore-Assisted

Carbohydrate Electrophoresis (FACE)

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Disaccharides Released byChondroitinase:

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Paper Chromatography of Released Disaccharides

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Thin-Layer Chromatography of Released Disaccharides

Silica Gel 60 TLC aluminum plate and developed with a solventsystem consisting of n -butanol/formic acid/water (4:8:1,).

Attaching a Chromophore for Analysis:

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Sigma Chemical Company

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HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB

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Separation of AMAC-Labeled Disaccharides by RP-HPLC:

HS

CS

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Disaccharides from Rat Liver GAGs-SAX-HPLC

HeparinasesCS-ABCase

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GAG Disaccharides from MDCK Cells

Heparinase

CSase ABC

2-aminobenzamide (2AB) labeled Disaccharides on Anion Exchange Columns:

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STDs Brain

CartilageSkin

Anion-Exchange Analysis of Linkage Region:

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Linkage Region

Scheme for Sequencing CS:

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FACE Analysis of Disaccharides

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FACE Analysis of GAG-Derived Disaccharides:

Identifying the Non-Reducing Ends

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Using Mercuric Acetate to IDReducing Ends:

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Using FACE to Analyze Non-Reducing Ends:

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Specific Enzymes to Confirm Sulfation:

Specific Enzymes to Confirm Sulfation:

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Biosynthesis of Chondroitin Sulfate

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NATURE CHEMICAL BIOLOGY | VOL 7 | NOVEMBER 2011

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. 53

MS/MS FT-ICR-MS CS GAG

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NATURE CHEMICAL BIOLOGY | VOL 7 | NOVEMBER 2011

Complete conversion to Na salt

FT-ICR negative-ion mass spectrum of 5.80-kDa MR fractionby PAGE with 18 isobars and 63 parent ions.

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NATURE CHEMICAL BIOLOGY | VOL 7 | NOVEMBER 2011

Deconvolution

CID-FT-ICR-MS/MS spectra of parent-ion m/z = 917.38

Sequencing by CID

56The proteoglycan bikunin has a defined sequenceMellisa Ly, Franklin E Leach III, Tatiana N Laremore, Toshihiko Toida, I Jonathan Amster & Robert J LinhardtNature Chemical Biology 7, 827–833 (2011) doi:10.1038/nchembio.673

Flow Chart for Analysis:

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. 57