Glycan and Monosaccharide Workshop Eoin Cosgrave David Wayland Bill Warren

Transcription

1 Glycan and Monosaccharide Workshop Eoin Cosgrave David Wayland Bill Warren 2012 Waters Corporation 1

2 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

3 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

4 Sample Preparation 2012 Waters Corporation 4

5 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

6 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

7 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

8 Chemistries for Glycan Analysis 2012 Waters Corporation 8

9 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

10 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

11 A focus on HILIC-FLR Glycans 2AB Total Analysis Glycoprotein PNGase F 2012 Waters Corporation 11

12 Converting Retention Times To GU Values 2012 Waters Corporation 12

13 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

14 GPC Option Installed in Empower 2012 Waters Corporation 14

15 Add GU to Project Custom Field 2012 Waters Corporation 15

16 GU = MP/10, Waters Corporation 16

17 Adjusting Fit Type and Calibration Order in Processing Method 2012 Waters Corporation 17

18 Alter the Dextran and Edit the Amount 2012 Waters Corporation 18

19 Enter Values for Each Peak in Dextran Ladder 10,000 to 150,000 (HPLC) 40,000 to 150,000 (UPLC) 2012 Waters Corporation 19

20 Run Dextran as a Narrow Standard Dextran n EU Minutes 2012 Waters Corporation 20

21 Waters Sells 2-AB Dextran Dextran n EU Minutes 2012 Waters Corporation 21

22 Calibrate Dextran Using Pre- assigned Values 2012 Waters Corporation 22

23 Integrate the Dextran Peaks 2012 Waters Corporation 23

24 Select Process Calibrate: Retention Times Shift to GU Values 2012 Waters Corporation 24

25 2012 Waters Corporation 25

26 For Samples, Integrate Peaks of Interest 45.0 EU EU Minutes 2012 Waters Corporation 26

27 Select Process Quantitate: Retention Times Shift to GU Values 45.0 EU EU Minutes 2012 Waters Corporation 27

28 GU and Peak Area Data Can Be Exported For Interrogation 45.0 EU RT % Area GU EU Waters Corporation 28

29 GlycoBase Waters Corporation 29

30 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

31 Glycan Structural Assignments Using Exoglycosidase Arrays 2012 Waters Corporation 31

32 Exoglycosidases Assist in Structural Elucidation JBM BTG SPG ABS BKF GUH NAN1 AMF CBG 2012 Waters Corporation 32

33 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 Any structure Bisecting GlcNAc β(1,4) α-mannose Galactose /β(1,3) /β1,4) Any structure NeuAc (2,3) Gal of any structure ~0.7 NeuAc (2,6) Gal of any structure ~ Waters Corporation 33

34 Structures are Identified by Mapping GU Shifts Murine IgG = x sialic acid = Tosoh 5 mm TSKgel Amide-80 (4.6 mm x 250 mm) 3 h Gradient Method Ex : 330 nm Em : 420 nm x 5.9 1x 2x = x galactose = x = x fucose = x = x GlcNAc = x man = x mannose = Waters Corporation 34

35 Specific Examples 2012 Waters Corporation 35

36 Identifying Galactose- (1-3) Galactose GU Value EU * * * * * * * * * * EU Retention Time (min) 2012 Waters Corporation 36

37 Determining LacNAcs GU Value A A3 6.0 A EU A1 M5 A4 M6 A3Lac1 M7 A2Lac2 Rh % e %\ W c B 1x g! M x g! x 4x g! g! EU x a%\! 2x a%\! Rh % e %\ W cg! Retention Time (min) 2012 Waters Corporation 37

38 Reference Material 2012 Waters Corporation 38

39 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/ Database and software: free for academics; can be licensed for commercial use Waters Corporation 39

40 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