Oligosaccharide Analysis Using High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection

Oligosaccharide Analysis Using High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection

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  1 Oligosaccharide Analysisby High-Performance Anion- Exchange Chromatography with Pulsed Amperometric Detection Jeff Rohrer, Ph.D. Director, Applications Development, Dionex Products The world leader in serving science
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  Agenda 2 •Introduction to HPAE-PAD • Carbohydrate analysis by HPAE-PAD • Fundamentals of HPAE-PAD oligosaccharide analysis • HPAE-PAD oligosaccharide analysis applied to glycoproteins • Comparison to other techniques and other topics • Conclusions
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  3 HPAE-PAD High-PerformanceAnion-Exchange Chromatography with Pulsed Amperometric Detection
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  HPAE-PAD: An EstablishedTechnique 4 • First described in the early 1980s • First applied to glycoproteins in 1986-1987 • Well over 1000 peer-reviewed publications using HPAE-PAD • Used in >10 United States Pharmacopeia official monographs
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  Basics of HPAE 5 • Carbohydrates are separated as oxyanions at high pH (>12). • These separations require hydroxide eluents. • If the carbohydrate is charged, acetate or another strong eluent must be added to the hydroxide eluent to elute the carbohydrate.
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  Basics of PAD 6 • Carbohydrates are detected on a gold working electrode (WE) at high pH by PAD. • PAD applies a series of potentials (a waveform) to a WE and the carbohydrate is detected by its oxidation at 1 potential. • The waveform is applied at a frequency of 2 Hz, i.e. two times a second.
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  Glycoprotein Monosaccharides 7 0 2 4 6 8 10 100 50 0 nC Minutes 1 2 3 4 5 6 Column: Thermo Scientific™ Dionex™ CarboPac™ PA20 Eluent: 10 mM Sodium hydroxide Temp: 30 ºC Flow Rate: 0.5 mL/min Inj. Volume:10 μL Detection: PAD (Waveform A TN21) Peaks: 1. Fucose 100 pmol 2. Galactosamine100 3. Glucosamine 100 4. Galactose 100 5. Glucose 100 6. Mannose 100
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  8 What isRequired for HPAE-PAD? • A non-metallic high-pressure liquid chromatography system • High-quality deionized water • Autosampler capable of injecting low microliter volumes • High-performance anion-exchange column designed for carbohydrates • Electrochemical detector and cell capable of executing the waveforms used for carbohydrate determinations
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  Why HPAE-PAD isan Established Technique 9 • No sample derivatization required for separation or detection – Direct detection • Sensitive detection • High-resolution separations • High capacity – can observe a small amount of one carbohydrate in the presence of a large amount of other • One technique able to determine a wide range of carbohydrates • One separation reveals both products and substrates – including labeled products
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  Popular HPAE-PAD Applicationsfor Glycobiology 10 • Monosaccharide compositional analysis • Sialic acid compositional analysis • Mannose-6-phosphate analysis • Glycoprotein oligosaccharide analysis
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  General HPAE-PAD Conditionsfor Glycoprotein Oligosaccharide Analysis 11 Column: Dionex CarboPac PA200 (3 x 250 mm) or Dionex CarboPac PA100 or Dioenx CarboPac PA1 (4 X 250 mm)* Eluent: 100 NaOH with a sodium acetate gradient up to as high as 500 mM Flow Rate: 0.5 mL/min (1.0 mL for PA1/100) Detection: PAD (Au) (See Technical Note 21) Samples: In water *9 X 250 mm and 22 X 250 mm have been used for preparative work.
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  Basic Thermo ScientificDionex CarboPac Construction 12 SO– SO– SO– SO– 3 3 3 3 SO– 3 SO– 3 SO– 3 Core Ion- Exchange Surface R3N+ R3N+ N+R3 N+R3 N+R3 R3N+ R3N+ N+R3 N+R3 R3N+ R3N+ N+R3 N+R3 R3N+ R3N+ N+R3 N+R3
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  Glycoprotein Oligosaccharide Analysis:Fetuin N-Linked Oligosaccharide Alditols 13 Column: Dionex CarboPac PA100 and guard Eluent: Sodium acetate gradient in 100 mM Sodium hydroxide Flow Rate: 1.0 mL/min Detection: Pulsed amperometry, gold electrode Sample: Fetuin oligosaccharide alditol standard Peaks: 1. Disialylated, triantennary 2. Disialylated, triantennary 3. Trisialylated, triantennary 4. Trisialylated, triantennary 5. Tetrasialylated, triantennary 6. Tetrasialylated, triantennary 7. Trisialylated, triantennary 1 2 3 4 5 6 7 0 10 20 30 40 50 50 nC 5 Minutes
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  Sialylated Oligosaccharide LinkageIsomer Separation 14 Neu5Ac(a2,6)Gal(b1,4)GlcNAc(b1,6) Neu5Ac(a2,6)Gal(b1,4)GlcNAc(b1,2)Man(a1,6) Man(b1,4)GlcNAc(b1,4)GlcNAc Neu5Ac(a2,3)Gal(b1,4)GlcNAc(b1,2)Man(a1,3) Neu5Ac(a2,3)Gal(b1,4)GlcNAc(b1,6) Neu5Ac(a2,6)Gal(b1,4)GlcNAc(b1,2)Man(a1,6) Man(b1,4)GlcNAc(b1,4)GlcNAc Neu5Ac(a2,3)Gal(b1,4)GlcNAc(b1,2)Man(a1,3) 3 4
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  Highly Sialylated OligosaccharideAlditols 110 nC 15 Column: Dionex CarboPac PA200 (3 x 250 mm) Guard (3 x 50 mm) Temp.: 30 C Flow: 0.5 mL/min Detection: PAD, Au on PTFE electrode Waveform: Carbohydrate (standard quad) Inj. Volume: 20 μL (full loop) Samples: Fetuin oligosaccharide alditol standard 1.5 10 30 50 70 85 20 Minutes Disialylated oligosaccharides Trisialylated oligosaccharides Tetrasialylated oligosaccharides
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  HPAE-PAD of GlycoproteinOligosaccharides 16 • Oligosaccharides must first be released from the protein. For asparagine (N-linked) oligosaccharides – PNGase F • For serine/threonine oligosaccharides – Reductive β-elimination. • Some samples can be directly injected into the HPAE-PAD system
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  General Conditions forPNGase F Digestion • 20-30 mg glycoprotein in 100-mL 50mM Sodium phosphate pH 7.6 10mM EDTA, 0.15% Triton X-100 (reduced) and 1 unit glycerol-free PNGase F. • Include 10mM beta-mercaptoethanol (BME), if needed. • Include 10mM BME and 10mM SDS, if needed. • Inject less than 50-mL. • BME can be removed by drying the sample. • SDS can be removed by cold EtOH precipitation. • REVIEW OF CONDITIONS FOR HPAE-PAD in AU176 17
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  Enzyme Cleavage Sites–PNGase F & Endos F2 and H 18 Man – GlcNAc – GlcNAc – Asn Man Protein Man Protein Endo F2 or Endo H PNGase F
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  General Conditions forBeta-Elimination* • Treat 20 mg protein with 100 mL1N NaBH4 in 0.1N NaOH. • Incubate for 24 h at 37 oC. • Neutralize with acetic acid. • Desalt (BioGel P2, 0.7 x 10 cm). 19 * - Anderson, D.C. et al. (1994) Glycobiology 4, 459-467.
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  Screening Total andCharged Oligosaccharides 60 nC 3.7 20 40 60 40 nC 20 Columns: Dionex CarboPac PA200, 3  250 mm and Dionex CarboPac PA200 Guard 3  50 mm Sample: PNGase F digestion of human transferrin. 28 B) Minutes 15 A) 3.7 20 40 60 Minutes Monosialylated glycans Disialylated glycans Monosialylated glycans Disialylated glycans AN 1050
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  21 28 19 B) B) 10 20 30 40 50 Minutes nC 1 2 3 4 6 5 8 7 Inj. Vol.: 5 μL Temp.: 30 °C Samples: A) PSA glycans after PNGase F release detergent removal, equivalent of 1 μg of protein injected, B) PNGase F control sample Peaks: 1. G0F 2. G1F isomers 3. G1 isomers 4. G2F 5. G2bF 6. A1F isomers 7. charged glycans 8. A2F isomers A) A) Monosialylated glycans Disialylated glycans Low Level Oligosaccharide Analysis
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  Low Level OligosaccharideAnalysis -Charged nC 22 C) B) 3.7 20 40 60 31 18.5 Minutes A) Monosialylated glycans Disialylated glycans Neutral glycans 1,2 3 4 5 Columns: Dionex CarboPac PA200, 3  250 mm and Dionex CarboPac PA200 Guard 3  50 mm Eluent: A) 100 mM NaOH B) 1M Sodium Acetate in 100 mM NaOH Gradient: 20-150 mM Sodium Acetate in 100 mM NaOH from 0- 75 min, 20 mM Sodium Acetate in 100 mM NaOH from 75.1-100 min Flow Rate: 0.5 mL/min Inj. Vol.: 5 μL Temp.: 30 °C Samples: A) Neutral glycan standards, B) PSA glycans after PNGase F release detergent removal, equivalent of 1 μg of protein injected, C) Transferrin glycans after treatment as with PSA glycans, equivalent of 1.3 μg of protein injected Peaks: 1. G0 2. G1F isomers 3. Man6 4. G2bF 5. Man9 AN 1050
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  23 Profiling andUsing Exoglycosidases To Assist in Identification of Structures
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  N-Linked Oligosaccharides fromThree IgG Preparations 24 Minutes 150 nA 25 190 nA 25 35% 49% 35% 10% 19% 14% 28% A B 1 2 3 4 1 2 3 5 0 10 20 30 40 50 300 nA 25 44% 37% 10% C 1 2 3 Column: Dionex CarboPac PA-100 Eluent: 0 to 250 mM NaOAc over 110 min in 100 mM NaOH Flow Rate: 1 mL/min Detector: Pulsed amperometry (Gold) Chromatograms: A. PNGase F digest of human polyclonal IgG B. PNGase F digest of murine MAB MY9-6 C. PNGase F digest of humanized MAB M115
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  Profiling N-linked Oligosaccharidesfrom hIgG and a MAb 25 Column: Dionex CarboPac PA 200 with guard Eluents: A 100 mM NaOH B 50 mM NaOAc in 100 mM NaOH C H2O D 0.5 M NaOAc in 100 mM NaOH Gradient: Time Flow rate %A %B %C %D (min) (ml/min) -10 0.35 48% 2% 50% 0% 0 0.35 48% 2% 50% 0% 50 0.35 37% 13% 50% 0% 51 0.35 45% 0% 50% 5% 80 0.35 24% 0% 50% 26% 81 0.45 0% 0% 0% 100% 91 0.45 0% 0% 0% 100% 92 0.45 48% 2% 50% 0% 100 0.45 48% 2% 50% 0% Temp: 30 °C Injection Volume: 10 μL Sample Conc.: ~5 μM Peaks: 1. G0F 2. Man5 3. G0 4,5. G1F 6. Man6 7. G2F 8. A1F 9. A2F MAb human IgG 2 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 min nC 3 2 1 standard mix 1 3 4 5 6 7 8 9 Zheng, T., Rohrer, J., Rao, S. (2010) Genetic Engineering News 30(10), 42-43.
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  Common N-linked Oligosaccharidesfound on IgG 26 N-acetylglucosamine (GlcNAc) Fucose (Fuc) Mannose (Man) Galactose (Gal) N-acetylneuraminic acid (Neu5Ac) Glycan (Oxford) mAb acronym Structure NGA2F (FA2G0) G0F NA2G1F (FA2G1) G1F NA2F (FAG2) G2F NA2FB (FABG2) G2bF G2FA1 (FA2G2S1) A1F G2FA2 (FAG2S2) A2F NGA2 (A2G0) G0 Man3 M3 Man5 M5 Man6 M6
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  nC 27 G1Fafter digestion G1F before digestion G0F standard 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 min 3 2 1 Column: CarboPac PA 200 with guard Gradient: 0-5 mM NaOAc in 50 mM NaOH from 0-40 min. Flow rate: 0.5 mL/min Temperature: 30 °C Injection Volume: 15 μL Sample Conc.: ~5 μM Samples: 1. G0 2. G1 3. G1 after digestion G1F Treated with Beta-Calactosidase
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  Monitoring Sialic AcidRelease from hlgG N-Linked Oligosaccharides 145 28 Column: CarboPac PA200 and guard Eluent: 0-5 min: 100 mM NaOH/5 mM NaOH 60 min: 100 mM NaOH/180 mM NaOH Temperature: 30 °C Flow Rate: 0.5 mL/min Injection Vol: 9 mL from 10-mL loop Detection: PAD (Au) Waveform A (TN 21) Sample: (1) PNGase-F digest of human IgG (2) Sialic acids standard (3) Neuraminidase digest (1) [nC] N-acetylneuraminic acid 0 5 10 15 20 25 30 15 3 2 1 [min] N-glycolylneuraminic acid
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  Degalactosylated N-Linked Oligosaccharidesfrom h.IgG 145 29 Column: CarboPac PA200 and guard Eluent: 0-5 min: 100 mM NaOH/5 mM NaOAC 60 min: 100 mM NaOH/180 mM NaOAc Temperature: 30 °C Flow Rate: 0.5 mL/min Injection Vol: 9 mL from 10-mL loop Detection: PAD (AU) Waveform A (TN21) Sample 1: PNGase-F of human IgG Sample 2: b-galactosidase digest of sample 1 0 20 40 15 1 2 nC min
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  Desialylated-Degalactosylated hIgG Oligosaccharides 150 30 Column: CarboPac PA200 and guard Eluent: 0-5 min: 100 mM NaOH/5 mM NaOAC 60 min: 100 mM NaOH/180 mM NaOAc Temperature: 30 °C Flow Rate: 0.5 mL/min Injection Vol: 9 mL from 10-mL loop Detection: PAD (AU) Waveform A (TN21) Sample 1: human IgG digested in turn by PNGase-F and neuraminidase Sample 2: b-galactosidase of sample 1 0 5 10 15 20 25 30 35 40 20 2 1 nC min
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  Empirical Relationships ofN-Linked Oligosaccharide Structure to Retention on the Dionex CarboPac PA1, Dionex CarboPac PA100, and Dionex CarboPac PA 200 Columns 1. The larger the oligosaccharide, the later it elutes. (e.g. the greater the 31 number of mannose residues in a high-mannose oligosaccharide, the later it elutes.) 2. Increased negative charge (sialylation, phosphorylation, sulfation), increased retention. 3. Addition of fucose to an oligosaccharide reduces its retention. 4. An oligosaccharide with a Neu5Aca23Gal elutes later than the same oligosaccharide with a Neu5Aca26Gal. 4 of 12 Rules from Rohrer, J. (1995) Glycobiology 5, 359-360.
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  32 Polysialic Acids
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  Separation of ColominicAcid with an Acetate Gradient 33
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  Separation with aNitrate Gradient (Stronger Eluent) 34
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  35 What AboutElectrochemical Response?
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  Tri-S 4B Tri-S2A Tri-S 6B Tri-S 5C β N-acetylglucosamine (GlcNAc) Mannose (Man) Galactose (Gal) N-acetylneuraminic acid (Neu5Ac) Tri-S 5D Tri-S 2B Tri-S 3D Linkages 6 4 3 1 2 1 α
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  Tetra-S 5 Tetra-S3 Penta-S 6
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  7 6 5 4 3 2 1 0 HPAE-PAD Peak Area Response of Fetuin Oligosaccharides Tri-S 4B Tri-S 2A Tri-S 6B Tri-S 5D Tri-S 5C Tri-S 2B Tri-S 3D Tetra-S 5 Tetra-S 3 Penta-S 6 Pak Area Respone (Glucose = 1) Fetuin Oligosaccharide Townsend, R. R.; et al. Anal. Biochem. 1988, 174, 459–470.
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  A B C -ol -ol -ol -ol D -ol E -ol Linkages 6 4 3 1 β N-acetylglucosamine (GlcNAc) Mannose (Man) Galactose (Gal) N-acetylneuraminic acid (Neu5Ac) N-acetylgalactosamine (GalNAc) N-glycolylneuraminic acid (Neu5Gc) F -ol G -ol H -ol I -ol J Sugars 2 1 α
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  1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 HPAE-PAD Peak Area Responses of O-Linked Oligosaccharides A B C D E F G H I J Peak Area Response (Oligosaccharide Alditol B=1.0) O-Linked Oligosaccharide Alditol Hayase, T.; et al. Anal. Biochem. 1993, 211, 72–80.
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  41 Purifying foror Interfacing to Mass Spectrometry
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  The Carbohydrate MembraneDesalter • Simple device automates the removal of sodium hydroxide and sodium acetate • Combines acidic regenerant and electrolysis to replace Na+ with hydronium ions • Delivers oligosaccharides in a weak acetic acid solution • No dialysis required 42
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  In-line Carbohydrate Desaltingwith the Carbohydrate Membrane Desalter 43 Eluent NaOAc and NaOH Sample O1- , O2- , O3- Analytical Column Amperometry Detector NaO1, NaO2, NaO3 in NaOH and NaOAc Regenerant Waste Acid Regenerant CMD-I HO1, HO2, HO3 in H2O and HOAc O1, O2, O3 = Oligosaccharides
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  44 Brief Comparisonto Other Techniques
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  HPAE-PAD Compared toMALDI-TOF • Field, M.; et al.Anal. Biochem. 1996, 239, 92–98. – quantification of oligosaccharides with both techniques yielded similar values. • Zhou, Q.; et al. Anal. Biochem. 2004, 335, 10–16. – Found that the two techniques yielded similar results • Gray, J. S.; et al. Glycobiology 1996, 6, 23–32. – Again found the two techniques yielded similar results except for isomers • Grey, C.; et al. J. Chromatogr., B 2009, 877, 1827–1832. – Relative glycan response similar for both methods. 45
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  HPAE-PAD Oligosaccharide AnalysisCompared to HILIC-FLD and CE-LIF • HPAE-PAD is orthogonal to CE-LIF. 46 • HPAE-PAD has some orthogonality to HILIC-FLD. • Working sensitivity is similar. • HPAE-PAD requires no sample derivatization. • HPAE-PAD may require more time per analysis. • HPAE-PAD has better separation of charged oligosaccharides. • HPAE-PAD preserves the oligosaccharides sialylation.
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  Other Carbohydrate Analytes– HPAE-PAD • Sugar phosphates, including Man-6-P, small inositol 47 phosphates • Sugar alcohols, e.g. mannitol • Small polysaccharides, e.g. maltodextrins to DP50 • Sulfated sugars, e.g. Gal-3-S • Sugar acids, e.g. galacturonic acid • Glycolipid oligosaccharides, including GPI anchors • Aminoglycosides (e.g. neomycin) • Sucralose • Fluorodeoxyglucose • Nucleotides and Nucleosides
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  Samples Analyzed byHPAE-PAD 48 • Glycoproteins, glycopeptides, glycolipids • Cane sugar • Serum and urine • Fermentation broths • Enzymatic reactions • Foods and beverages • Vaccines, pharmaceutical preparations • River and sea waters • Wood pulp
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  Conclusions • HPAE-PADprovides high resolution separations of both N-linked 49 and O-linked oligosaccharides derived from glycoproteins. • The same HPAE-PAD system can be used for determinations of monosaccharides, sialic acids, sugar phosphates, and other carbohydrates. • HPAE-PAD can be used to prepare pure oligosaccharides from NMR or MS, or can be interfaced directly with MS.
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  50 Thank youfor your attention! WS71459-EN 1214S