6Examples of keratan sulfate proteoglycans Essentials of Glycobiology
7Examples of heparan sulfate proteoglycans Essentials of Glycobiology
8Proteoglycan Analysis Isolation and Analysis of Intact ProteoglycansIdentification of Core Protein – Sequence.Site-Mapping of Proteoglycans & Other PTMsIsolation & Characterization of GlycosaminoglycansDigestions to Produce disaccharide repeatDetermining Repeat CompositionSulfationNon-reducing terminusSequencing of GAGs
9Use of Denaturing Chaotropic Agents to Isolate Proteoglycans:
10Isolation of Intact Proteoglycans 35SO4 + 3H-glucosamineProteoglycan ProtocolsEdited by Renato V. Iozzo, MD
11Typical Work Flow - Proteoglycans Extraction of Proteoglycans – Typically 4M Urea or 6M GuHCl –Strong denaturing/chaotropic agents.Membrane bound PGs require DetergentsIon-Exchange -DEAE-Sephacel or similar anion exchange enrichment. – High negative charge.Size Exclusion Chromatography – Typically Sepharose 4BAnalysis of GAG chains after release –Protease DigestionBeta-EliminationGAG size fractionation – TSK4000, HPLC, Sephadex G200, Superose CL-6B
12GPs PGs Negative Charge Allows Ready Separation from Other Glycans Proteoglycan ProtocolsEdited by Renato V. Iozzo, MD
13Size Fractionation of Proteoglycans Proteoglycan ProtocolsEdited by Renato V. Iozzo, MD
19Analysis of Glycosaminoglycans Release from PGs – Protease, Beta-EliminationLyases & Hydrolases – Fragment GAGsDisaccharide Compostion AnalysisSulfation SitesNon-Reducing TerminusMercuric acetate elimination of unsaturated bond containing disaccharides reveals non-reducing TerminusPresence of Classical N- & O-GlycansLinkage of oligosaccharidesO-Glycans in beta-eliminated GAGS
20GAG Degrading Enzymes: Hydrolases & Eliminases: Hydrolase – Catalyzes Hydrolysis i.e. Addition of water across a chemical bond.A-B + H2O A-OH + B-HExamples: testicular Hyase; endo-b-galactosidaseEliminase – Catalyzes the removal of H2O from a chemical bond.A-B A-OH + dB + H2OExamples: chondroitinase ABC, HS lyases, Strept. Hyaluronidase.
21Characterization of Glycosaminoglycans: Bacterial EliminasesArePowerful Tools:SequentialDegradationFollowed byGel Filtration.2. Other SeparationMethods.
22Degrades All Chondroitin Sulfates, Dermatan Sulfates and Hyaluronic Acid
23Digests All Types of Chondroitin Sulfates and Hyaluronic Acids, but Not Digest Hyaluronic Acids.
36Thin-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,).
37Attaching a Chromophore for Analysis: Sigma Chemical Company
38HPLC separation of CS-derived saturated and unsaturated disaccharides labeled with 2ABFIG. 2. HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB. CS-derived saturated tetrasaccharidesTetra-C1–C4 were individually digested with chondroitinaseAC-II and derivatized with 2AB. The four digests and 2ABderivatizedunsaturated CS-disaccharides were mixed and analyzedby HPLC as described in the legend to Fig. 1. The HPLC conditionswere the same as those for the experiments shown in Fig. 1.
39Separation of AMAC-Labeled Disaccharides by RP-HPLC: HSCS
40Disaccharides from Rat Liver GAGs-SAX-HPLC HeparinasesCS-ABCase
41GAG Disaccharides from MDCK Cells HeparinaseCSase ABC
422-aminobenzamide (2AB) labeled Disaccharides on Anion Exchange Columns: STDsBrainCartilageSkin
52Biosynthesis of Chondroitin Sulfate NATURE CHEMICAL BIOLOGY | VOL 7 | NOVEMBER 2011
53Difficulties 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 sequencingdifficulties 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.
54MS/MS FT-ICR-MS CS GAG Complete conversion to Na salt NATURE CHEMICAL BIOLOGY | VOL 7 | NOVEMBER 2011
55FT-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.DeconvolutionCID-FT-ICR-MS/MS spectra of parent-ion m/z =fraction. (a) FT-ICR negative-ion mass spectrum of 5.80-kDa MR fractionby PAGE with 18 isobars and 63 parent ions. (b) Deconvolution ofspectrum a. (c) CID-FT-ICR-MS/MS spectra of parent-ion m/z =(z = 6) and annotated fragment-ions providing sequence with dp27-5-Serfragmentation pattern assigned from spectrum.Sequencing by CIDNATURE CHEMICAL BIOLOGY | VOL 7 | NOVEMBER 2011
56Flow 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 sequenceMellisa Ly, Franklin E Leach III, Tatiana N Laremore, Toshihiko Toida, I Jonathan Amster & Robert J LinhardtNature Chemical Biology 7, 827–833 (2011) doi: /nchembio.673
57Analysis 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.