Glycan and Monosaccharide Workshop Eoin Cosgrave David Wayland Bill Warren 2012 Waters Corporation 1
Requests and Questions Optimised sample prep protocol to reduce sample preparation time How can I detect glycans with an PDA, is it possible to detect them without labeling? Is a HILIC column the best choice? How can I detect glycans with an PDA, is it possible to detect them without labeling? Is a HILIC column the best choice? Very interested to determine specially attached to nano-particles Which dextran ladder use? How to convert RT to GU? How to use glycobase 3+ Want an overview Would it be possible to separate and detect correctly sialylated N-Glycans from nonsialylated main N-Glycans in the same analysis when using UPLC HPLC vs MALDI Tof Overview of subject High throughput sample preparation and standards Automatisation for preparation sample 2012 Waters Corporation 2
Workshop Overview What we will cover Sample Preparation Chemistries for Glycan Analysis Generating GU Values Using GlycoBase 3.1 The Exoglycosidases Practical Glycan Structural Assignments But before we begin How familiar are you with glycan analysis? Are you currently performing glycan analysis? Are you familiar with the Waters solution for glycan analysis? Are you interested in other types of glycans? Do you use Empower for analysis? How familiar are you with the process for integrating glycan separations? What methods do you currently use? What are your existing and expected challenges? Do you perform monosaccharide or sialic acid analysis? If so, how? 2012 Waters Corporation 3
Sample Preparation 2012 Waters Corporation 4
In-Gel versus In-Solution In-Gel PNGase F 1) Immobilization (SDS-PAGE) 2) Reduction (DTT) 3) Alkylation (IAA) 4) Buffer Exchange (H 2 O/20 mm NaHCO 3 ) 5) PNGase F Digestion 6) Released Glycan Recovery (H 2 O/MeCN) 7) Centrifugal Evaporation 8) Formic Acid Treatment 9) Centifugal Evaporation 10) 2-AB Labelling 11) Removal of Excess 2-AB (NP tips) 12) Centrifugal Evaporation 13) Reconstitution (H 2 O) Total 30 min 45 min 20 min 2 h O/N* ~3 h ~5 h 40 min ~1 h 2 h to O/N 30 min ~3 h 2 d 17 h 2012 Waters Corporation 5
In-Gel versus In-Solution In-Solution PNGase F 1) Reduction (DTT) 2) Alkylation (IAA) 3) Buffer Exchange (MWCO filters) 4) PNGase F Digestion 5) Released Glycan Recovery (MWCO Filters) 6) Glycan Desalting (PGC SPE) 7) Centrifugal Evaporation 8) Formic Acid Treatment 9) Centrifugal Evaporation 10) 2-AB Labelling 11) Removal of Excess 2-AB (NP tips) 12) Centrifugal Evaporation 13) Reconstitution (H 2 O) Total 45 min 20 min 20 min O/N* ~30 min ~30 min ~2 h to O/N 40 min ~1 h 30 min ~3h 2 d 12 h 2012 Waters Corporation 6
Pros and Cons Pro Con In-Gel 1) Buffer compatibility 2) Proven high throughput capacity 3) Compatible with automated liquid handlers 1) Sample amounts limited to mg quantitites 2) Time intensive preparation In-Solution 1) Larger amounts of sample possible (mg amounts) 2) Quicker and easier sample processing 3) Removal of gel peaks in analysis 1) Cost of enzyme used 2) Not compatible with all glycoproteins Note: kits/labels now available that accelarate the sample prep process 2012 Waters Corporation 7
Chemistries for Glycan Analysis 2012 Waters Corporation 8
LC Separation Chemistries Characterize Glycan Attributes Hydrophilicity/size HILIC (Amide-80) Acquity Acquity Waters Waters Total glycan population (neutral/charged) Total glycan population relative quantitation High reproducibility High sensitivity RP (C18) hydrophobicity Acquity Waters Glycan separation based on attributes (fucose, bisects, mannose) High reproducibility High sensitivity 2AB-labelled Glycans WAX (DEAE) Increased Negative Charge Glycan separation based on charge (NeuAc, PO 4, SO 3 ) 2012 Waters Corporation 9
Different Labels Required for Different Analyses DMB NeuAc Speciation Glycans 2AB Total Analysis Charged Glycans Glycoprotein PNGase F Unlabelled Mass Spec Protein Digestion Proteomics 2012 Waters Corporation 10
A focus on HILIC-FLR Glycans 2AB Total Analysis Glycoprotein PNGase F 2012 Waters Corporation 11
Converting Retention Times To GU Values 2012 Waters Corporation 12
Brief Summary 1) Ensure GPC option is available in Empower 2) Add GU custom calculation to your project custom field 3) Adjust processing method fit type for 5 th order polynomial 4) Run a dextran as a Broad or Narrow Standard 5) Assign mass amounts to dextran ladder 6) Calibrate dextran 7) Integrate your sample of interest 8) Quantitate 2012 Waters Corporation 13
GPC Option Installed in Empower 2012 Waters Corporation 14
Add GU to Project Custom Field 2012 Waters Corporation 15
GU = MP/10,000 2012 Waters Corporation 16
Adjusting Fit Type and Calibration Order in Processing Method 2012 Waters Corporation 17
Alter the Dextran and Edit the Amount 2012 Waters Corporation 18
Enter Values for Each Peak in Dextran Ladder 10,000 to 150,000 (HPLC) 40,000 to 150,000 (UPLC) 2012 Waters Corporation 19
Run Dextran as a Narrow Standard 16.00 15.00 14.00 13.00 10000 Dextran 12.00 11.00 10.00 9.00 8.00 20000 n EU 7.00 6.00 5.00 4.00 3.00 2.00 1.00 30000 40000 50000 60000 70000 80000 90000 100000 110000 120000 130000 140000 150000 0.00-1.00-2.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00 120.00 130.00 140.00 150.00 160.00 170.00 180.00 Minutes 2012 Waters Corporation 20
Waters Sells 2-AB Dextran 46.0 44.0 42.0 40.0 38.0 36.0 34.0 Dextran 7 8 9 10 11 12 13 14 15 16 17 18 19 20 32.0 30.0 28.0 n 6 26.0 EU 24.0 22.0 5 20.0 18.0 4 16.0 14.0 12.0 10.0 8.0 6.0 4.0 2.0 0.0-2.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0 42.0 44.0 Minutes 2012 Waters Corporation 21
Calibrate Dextran Using Pre- assigned Values 2012 Waters Corporation 22
Integrate the Dextran Peaks 2012 Waters Corporation 23
Select Process Calibrate: Retention Times Shift to GU Values 2012 Waters Corporation 24
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For Samples, Integrate Peaks of Interest 45.0 EU 40.0 35.0 30.0 25.0 20.0 15.0 5.584 8.832 12.215 15.384 18.235 20.776 23.027 10.0 5.0 0.0 3.5 3.0 11.733 14.132 16.665 2.5 EU 2.0 1.5 1.0 0.5 0.0 9.904 10.370 12.498 12.933 13.220 14.484 15.032 15.271 15.557 16.237 17.253 18.075 18.958 19.892 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Minutes 2012 Waters Corporation 26
Select Process Quantitate: Retention Times Shift to GU Values 45.0 EU 40.0 35.0 30.0 25.0 20.0 15.0 40000 50000 60000 70000 80000 90000 100000 10.0 5.0 0.0 3.5 3.0 58917 66208 74512 2.5 EU 2.0 1.5 1.0 0.5 0.0 53538 54904 57183 61199 62514 63387 65565 67317 68005 69069 69845 70781 73053 76567 78203 78715 79529 82842 86500 87367 89266 98516 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Minutes 2012 Waters Corporation 27
GU and Peak Area Data Can Be Exported For Interrogation 45.0 EU 40.0 35.0 30.0 25.0 RT % Area GU 1 5.39 3.05 4 2 20.0 8.69 4.23 5 3 15.0 12.12 5.43 6 4 15.32 6.77 7 10.0 5 18.19 7.99 8 40000 6 20.74 5.0 9.07 9 7 23.01 9.95 10 0.0 8 25.04 10.56 11 9 26.85 3.5 10.86 12 10 28.50 10.95 13 3.0 11 30.01 10.85 14 12 31.39 10.29 15 2.5 50000 58917 60000 66208 70000 74512 80000 90000 100000 EU 2.0 1.5 1.0 0.5 0.0 53538 54904 57183 61199 62514 63387 65565 67317 68005 69069 69845 70781 73053 76567 78203 78715 79529 82842 86500 87367 89266 98516 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 2012 Waters Corporation 28
GlycoBase 3.1 2012 Waters Corporation 29
Brief Summary 1) An on-line repository of glycan structures 2) Structures provided in GU values 3) Multi-notation options (Oxford, CFG, text) 4) Data interpretation assistance (Exoglycosidases) 5) Data filtering options (sample- or structure-based) 2012 Waters Corporation 30
Glycan Structural Assignments Using Exoglycosidase Arrays 2012 Waters Corporation 31
Exoglycosidases Assist in Structural Elucidation JBM BTG SPG ABS BKF GUH NAN1 AMF CBG 2012 Waters Corporation 32
Characteristic GU Shifts Aid in Analysis Monosaccharide Linkage To GU Increment Core fucose (1,6) Any structure 0.5 Outer arm fucose (1,3) (1,6) GlcNAc 0.8 Outer arm fucose (1,2) Gal 0.5 Mannose GlcNAc (1,2) (1,3) (1,6) β(1,2) β(1,4) β(1,3) β(1,6) Man 0.7-0.9 Any structure 0.5-0.6 Bisecting GlcNAc β(1,4) α-mannose 0.1-0.15 Galactose /β(1,3) /β1,4) Any structure 0.8-0.9 NeuAc (2,3) Gal of any structure ~0.7 NeuAc (2,6) Gal of any structure ~1.15 2012 Waters Corporation 33
Structures are Identified by Mapping GU Shifts Murine IgG1 8.9-7.5 = 1.4 2 x sialic acid = 1.4 8.9 Tosoh 5 mm TSKgel Amide-80 (4.6 mm x 250 mm) 3 h Gradient Method Ex : 330 nm Em : 420 nm 6.7 7.5 2x 5.9 1x 2x 7.5 5.9 = 1.6 2 x galactose = 1.6 5.5 1x 5.9 5.5 = 0.4 1 x fucose = 0.4 4.4 2x 5.5 4.4 = 1.1 2 x GlcNAc = 1.1 2.7 2x man 4.4 2.7 = 1.7 2 x mannose = 1.7 2012 Waters Corporation 34
Specific Examples 2012 Waters Corporation 35
Identifying Galactose- (1-3) Galactose GU Value 6 7 8 9 10 11 12 13 14 15 16 EU 8.0 6.0 4.0 2.0 0.0 * * 9.46 9.66 * * * 10.01 10.47 * 11.26 10.78 * * * * 4.0 8.82 3.0 EU 2.0 8.42 1.0 0.0 80.0 85.0 90.0 95.0 100.0 105.0 110.0 115.0 120.0 125.0 130.0 135.0 140.0 Retention Time (min) 2012 Waters Corporation 36
Determining LacNAcs GU Value 2 3 4 5 6 7 8 9 10 11 12 13 14 15 A A3 6.0 A2 5.0 4.0 EU 3.0 2.0 1.0 0.0 A1 M5 A4 M6 A3Lac1 M7 A2Lac2 Rh % e %\ W c B 1x g! M3 20.0 2x g! 15.0 3x 4x g! g! EU 10.0 5.0 1x a%\! 2x a%\! Rh % e %\ W cg! 0.0 30 40 50 60 70 80 90 100 110 120 130 140 Retention Time (min) 2012 Waters Corporation 37
Reference Material 2012 Waters Corporation 38
Immobilized Glycoprotein Immobilized protein PNGase F Elute Wash Release Dry Formic Acid Dry Elute Wash Elute 2-AB Label 2AB-labelled Glycans Patent No: PCT/IB2005/002995 Database and software: free for academics; can be licensed for commercial use. 2012 Waters Corporation 39
In-Solution Release and Labelling! In-solution release does not work for all proteins! DTT IAA / IAM PNGase F Elute Recover Release Desalt (PGC SPE) Dry Elute Wash Elute Formic Acid 2-AB Label 2AB-labelled Glycans 2012 Waters Corporation 40