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Monosaccharide, Oligosaccharide, and Linkage Analysis Natasha E. Zachara Ph.D. nzachara@jhmi.edu

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O O HO AcNH What questions might you ask? What monosaccharides modify my protein? What oligosaccharides modify my protein? How are these sugars linked to each other? As always, the choices you make about how you will approach answering these questions will depend in part on the equipment available to you.

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O O HO AcNH Why? Knowing which sugars helps to predict the type of oligosaccharides present and thus helps determine the approach to detailed structural analysis It can also provide the first clues to the presence of a new type of sugar chain. Step one: release of sugars Step two: labeling of sugars (if necessary) Step three: detection and quantification Monosaccharide Analysis

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O O HO AcNH Sample Preparation In most cases, salt is an issue: Proteins can be precipitated to remove salt Acetone Methanol Methanol/Chloroform Protein can be desalted by Size Exclusion Chromatography Ammonium formate, ammonium bicarbonate (volatile) Sephadex-G50, many others Proteins/peptides purified by reversed phase are also acceptable Proteins can be separated by SDS-PAGE and blotted to PVDF Sugars can be desalted Size exclusion chromatography (time challenging) Dowex Ion Exchange (in a volatile buffer) Graphitized Carbon

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O O HO AcNH Release: monosaccharides Acid hydrolysis: Different sugars and linkages are released at different rates Low acid: Sialic acid Neutral and amino sugars: 2M TFA and 4M HCl Harazono AHarazono A, et al., A comparative study of monosaccharide composition analysis as a carbohydrate test for biopharmaceuticals. Biologicals. 2011 May;39(3):171-80. PMID: 21549615Biologicals.

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O O HO AcNH Detection The challenge is detecting your released sugars: PAD: Pulsed Ampometric Detection (no labeling) Metabolic Labeling Derivatization for HPLC: 2-aminopyridine (2AP), ethyl 4-aminobenzoate (ABEE), 2-aminobenzoic acid (2AA) 1-phenyl-3-methyl-5-pyr- azolone (PMP) 1,2-diamino-4,5-methylenedioxybenzene (DMB) Capillary Electrophoresis: 8-aminopyrene-1,3,6-trisulfonate (APTS) 2-aminoacridone derivatization Derivatization for GC-MS: trime- thylsilyl or alditol acetate Harazono AHarazono A, et al., A comparative study of monosaccharide composition analysis as a carbohydrate test for biopharmaceuticals. Biologicals. 2011 May;39(3):171-80. PMID: 21549615Biologicals.

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O O HO AcNH Separation HPLC Anion Exchange Chromatography (HPAEC) Reversed Phase Ion Exchange (TSK Fractogel) Capillary Electrophoresis GC-MS

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O O HO AcNH High Performance Anion Exchange Chromatography- Pulsed Ampometric Detection (HPAEC-PAD) Advantages: No Labeling No interference from labeling reagent Reasonable sensitivity (10pmol) Disadvantages: Specialized HPLC (All hydroxide) The Separation: Under alkaline conditions, the hydroxyl groups are ionized to oxyanions A monosaccharide possesses several ionizable hydroxyl groups with the following hierarchy of acidity: 1-OH > 2-OH >> 6-OH > 3-OH > 4-OH The varying location of the OH groups results in slight differences in the pK a value (ranging from 12 to 14) of individual monosaccharides Harazono AHarazono A, et al., A comparative study of monosaccharide composition analysis as a carbohydrate test for biopharmaceuticals. Biologicals. 2011 May;39(3):171-80. PMID: 21549615Biologicals.

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O O HO AcNH HPAEC-PAD Columns Behan JLBehan JL, Smith KD. The analysis of glycosylation: a continued need for high pH anion exchange chromatography. Biomed Chromatogr. 2011 Jan;25(1-2):39-46. PMID: 20821735Smith KDBiomed Chromatogr. CarboPAc PA1, 10, 20 Monosaccharides Disaccharides Sialic acid CarboPAc PA 100, 200 Oligosaccharides CarboPAc MA1 Aliditols (monosaccahrides, oligosaccharides)

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O O HO AcNH How does pad work? Ampometric Detection: Any analyte that can be oxidized or reduced is a candidate for amperometric detection A voltage (potential) is applied between two electrodes positioned in the column effluent The measured current changes as an electroactive analyte is oxidized at the anode. Pulsed Ampometric Detection: Important as it ensures that the gold electrode does not become coated by oxides In pulsed amperometric detection (PAD), a working potential is applied for a short time (usually a few hundred milliseconds), followed by higher or lower potentials that are used for cleaning the electrode The detection of monosaccharides and amine- containing glycoconjugates with low levels of glycosylation can be compromised from fouling of the working electrode by amino acids.

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O O HO AcNH Examples: monosaccharides http://www.dionex.com/

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O O HO AcNH Examples: monosaccharides http://www.dionex.com/ Note, that Sodium Acetate is required to elute sugar phosphates

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O O HO AcNH Sugar Alditols Note, that a gradient of sodium hydroxide is required http://www.dionex.com/

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O O HO AcNH HPAEC: SIALIC acid The strong charge requires more robust conditions; an increasing sodium acetate linear gradient is useful (50250 mm over 30 min. O-Acetyl groups can be labile, andcan saponify during analysis. There are alternative non-high pH based methods. http://www.dionex.com/

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O O HO AcNH HPAEC: Neutral oligosaccharides Separation of oligosaccharide is based on size, charge, monosaccharide composition and intra-chain linkages Separation is based on a sodium acetate gradient; the greater the negative charge (greater the number of NeuAc) and number of sugars, the higher the sodium acetate concentration required http://www.dionex.com/

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O O HO AcNH HPAEC: Charged Oligosaccharides the greater the overall negative charge, the greater the retention time. http://www.dionex.com/

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O O HO AcNH Desalting post-HPAEC Packer NHPacker NH, Lawson MA, Jardine DR, Redmond JW. Glycoconj J. 1998 Aug;15(8):737-47. A general approach to desalting oligosaccharides released from glycoproteins. PMID: 9870349Lawson MAJardine DRRedmond JWGlycoconj J.

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O O HO AcNH GC-MS: monosaccharides Sugars must be derivatized: trimethyl esters, aldlitol acetates, permethylation Also possible to release sugars by methanolysis, and derivatize to TMS derivatives Detection: Flame Ionization detector Several types of stationary phases can be used for the analysis of acetylated alditols: CP-Sil 5 WCOT, SP-1000, Silar 10C, OV-1, SE-54, 5% DB-5, DB-1, Carbowax 20M, SE-30, OV-101, and OV-275. Other stationary phases used for the analysis of trimethylsilylated glycosides and methyl glycoside methyl esters include CP-Sil 5 WCOT, 5% DB-5, DB-1, SE-30, and OV-101. PMID: 6057635

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O O HO AcNH GC-MS: monosaccharides Identify sugars by retention time and fragmentation patterns Lose some information re: modifications of sugars Quantitative PMID: 6057635

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O O HO AcNH HPLC: monosaccharides Labeling with different derivatives: A: 2AP (2-aminopyridine ) B: ABEE (ethyl 4-aminobenzoate ) C: 2AA (2-aminobenzoic acid ) D: PMP (1-phenyl-3-methyl-5-pyrazolone ) Separation Technologies: Strong Anion Exchange (TSK Fractogel) Reversed Phase (C18) CE Great sensitivity Derivatization can fluctuate in efficiency May be necessary to include a clean up step Longer run times Harazono AHarazono A, et al., A comparative study of monosaccharide composition analysis as a carbohydrate test for biopharmaceuticals. Biologicals. 2011 May;39(3):171-80. PMID: 21549615Biologicals.

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O O HO AcNH Capillary Electrophoresis (CE) Abo M, He LP, Sato K, Okubo A. Determination of monosaccharides derivatized with 2-aminobenzoic Acid by capillary electrophoresis. Methods Mol Biol. 2013;984:45-50. doi: 10.1007/978-1-62703-296-4_4. PMID:23386335 Separation buffers: 50 mM sodium phosphate (pH 5.5) or 150 mM sodium borate-50 mM sodium phosphate (pH 7.0) running buffer. Run times are fast Compatible with mass spectrometry Not preperative

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O O HO AcNH Oligosaccharides There are three steps: Step one: Release Step two: Detections Step three: Separation

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O O HO AcNH Release: oligosaccharides Reductive -elimination: releases O-linked sugars, but the carbohydrates must be reduced to prevent peeling. results in sugar alditols, which are not compatible with many labeling techniques Alditol acetates are volatile and are easily detected by GC-MS. Hydrazinolysis: Hydrazine hydrolysis is an effective method for the complete release of unreduced O- and N-linked oligosaccharides. not as popular as other techniques as its toxic and can affect the integrity of the peptide/protein backbone. Enzyme hydrolysis: PNGase F releases most N-linked glycans.

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O O HO AcNH Detection: Oligosaccharides Radiolabels: Metabolic Labeling Glycosyltransferases -elimination Reductive amination: Incorporation of fluoresecent derivatives (see above) Pulsed amometric detection Mass spectrometry (GC, MALDI, ESI-MS)

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O O HO AcNH Separation Thin Layer Chromatography High-voltage Borate Paper electrophoresis FACE Size exclusion/Gel Permeation Chromatography: HPLC Graphitized Carbon Reversed Phase HILIC HPAEC GC-MS Mass Spectrometry Can be combined with HPLC

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O O HO AcNH HPLC: Graphitized Carbon Not entirely clear how graphitized carbon separates oligosaccharides, although size, shape and charge are all important Can separate anomers N-linked sugars should be reduced to avoid peak splitting Sensitive to the low femtomole level Columns require significant regeneration Karlsson NG, Wilson NL, Wirth HJ, Dawes P, Joshi H, Packer NH. Rapid Commun Mass Spectrom. 2004;18(19):2282-92. Negative ion graphitised carbon nano-liquid chromatography/mass spectrometry increases sensitivity for glycoprotein oligosaccharide analysis.

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O O HO AcNH HPLC: hydrophilic interaction liquid chromatography (HILIC) HILIC: Sugars are separated by a partitioning mechanism in contrast to the traditional adsorption chromatography on normal phase materials Typical equilibration conditions 1025% water in acetonitrile with a low concentration of acid or salt (mostly below 100 mM) Common columns include: TSK Gel-Amide 80 and ZIC-HILIC-SPE Compatible with spectrometry Detection requires derivatization

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O O HO AcNH Biogel p4 Carbohydrates can be separated by size Typically Biogel P-2, P-4 and P-6 (

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O O HO AcNH FACE: fluorophore-assisted carbohydrate electrophoresis Separate oligosaccharides by electrophoresis Addition of a tag (by reductive amination) provides a charge and a meads of detection Separation may vary if sugars are sulfated, acetylated of sialylated as this will change the size: charge ratio. Can be combined with enzyme treatments to look at the structure of the oligosaccharides, however, this requires purified oligos

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O O HO AcNH GC-MS: Oligosaccharides Large oligosaccharides (>10) are difficult to detect Must use alternative approaches, eg. FAB-MS Typically permethylated Karlsson N G et al. J. Biol. Chem. 1997;272:27025-27034

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O O HO AcNH Fragmentation: Oligosaccharides Karlsson N G et al. J. Biol. Chem. 1997;272:27025-27034

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O O HO AcNH Linkage Analysis What questions are you really asking? Which sugars are linked to each other, and in what order Though which hydroxyl residues are these sugars linked What is the anomericity of the linkage? Methods: NMR (best, but requires largish amounts of pure material) Exoglycosidase digestion of oligosaccharides (requires the right enzymes) Methylation Analysis: GC-MS ESI-MS (best with upstream separation of oligosaccharides) You may get some information about anomericity when this is performed in tandem with column chromatography MALDI-TOF Mass Spectrometry

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O O HO AcNH Methylation Analysis Chapter 47, Figure 3 Essentials of Glycobiology Second Edition Methylation analysis is a well-established method for determining linkage positions. 1)Introduce a stable substituent (an ether-linked methyl group) onto each free hydroxyl group of the native glycan 2)Acid cleavage (glycosidic linkages and more labile than ether-linked methyl groups) 3)Partially methylated monosaccharides with free hydroxyl groups are derivatized to produce volatile molecules amenable to GLC-MS analysis. 4)Typically, monosaccharides are reduced (no alpha or beta anomers) followed by acetylation of the free hydroxyls 5)Detection by GC-MS or ESI-MS No sequence information, no anomeric linkage information

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O O HO AcNH Methylation Also Improves Assignment During Mass Spectrometry What Mass Spectrometry can do: 1) heterogeneity and type of glycosylation; 2) sites of glycosylation; 3) glycan-branching patterns; 4) the number and lengths of antennae, their building blocks, and the patterns of substitution with fucose, sialic acids, or other capping groups such as sulfate, phosphate, or acetyl esters; 5) complete sequences of individual glycans. What it cant do: Which Glucose versus Mannose Anomericity How does Methylation Help: Better data Greater confidence in linkage analysis by MS

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O O HO AcNH Wednesday This will be a demonstration laboratory: You will set up one protein for sequencing (and analyze the cycles) We will talk about strategies for site-mapping proteins (mini-lecture) You will analyze some spectra of O-GlcNAc modified peptides

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O O HO AcNH Reading Harazono A, et al., A comparative study of monosaccharide composition analysis as a carbohydrate test for biopharmaceuticals. Biologicals. 2011 May;39(3):171-80. PMID: 21549615 Harazono ABiologicals. Behan JL, Smith KD. The analysis of glycosylation: a continued need for high pH anion exchange chromatography. Biomed Chromatogr. 2011 Jan;25(1-2):39-46. PMID: 20821735 Behan JLSmith KDBiomed Chromatogr. Manzi AE. 2001 May;Chapter 17:Unit17.19A. Total compositional analysis by high-performance liquid chromatography or gas-liquid chromatography. PMID: 18265151 Manzi AE Manzi AE, Diaz S, Varki A. Anal Biochem. 1990 Jul;188(1):20-32. High-pressure liquid chromatography of sialic acids on a pellicular resin anion-exchange column with pulsed amperometric detection: a comparison with six other systems. PMID: 2221361 Manzi AEDiaz SVarki AAnal Biochem. Reinhold V, Zhang H, Hanneman A, Ashline D. Mol Cell Proteomics. Toward a platform for comprehensive glycan sequencing. 2013 Apr;12(4):866-73. PMID: 23438731 Reinhold VZhang HHanneman AAshline DMol Cell Proteomics. Hansson GC, Karlsson H. Methods Mol Biol. Gas chromatography and gas chromatography-mass spectrometry of glycoprotein oligosaccharides. 1993;14:47-54. PMID: 8348243 Hansson GCKarlsson HMethods Mol Biol. Kim JH, Shome B, Liao TH, Pierce JG. Anal Biochem. Analysis of neutral sugars by gas-liquid chromatography of alditol acetates: application to thyrotropic hormone and other glycoproteins. 1967 Aug;20(2):258-74. PMID: 6057635 Kim JHShome BLiao THPierce JG Anal Biochem.