1. Methods for High-Throughput Analysis of Protein Glycosylation Robotics Applications Assay Development

3. Glycoprotein affinity purification (e.g. IgG) Glycan preparation Glycan enrichment, labelling and clean-up UPLC / MS analysis Statistical data analysis Protein denaturation Alkylation Glycan release Glycan elution Glycan capture on solid support Integration and quantification Labelling clean-up 1 4 1 4 3 2 1 1 Washing & glycan release Fluorescent labelling Protein binding Washing & elution Solvent removal 2 3 2 3 Cell culture sample, serum Data analysis and visualisation 2 (1)Cell secretome glycomics (2)Whole serum glycomics (3)IgG glycomics (4)PGC cleanup Development of an automated glycomics platform Anal. Chem. 2013, 85, 8841-8849

4. IgG glycomics workflow – 2AB labelling Anal. Chem. 2013, 85, 8841-8849

5. Glycan sample clean-up using solid supports Anal. Chem. 2008, 80, 1094-1101

6. Importance of sample clean-up for 2AB labelling No clean-up

7. Development of an IgG N-Glycosylation Protocol Minutes 3.003.504.004.505.005.506.006.507.007.508.008.509.009.5010.0010.5011.0011.5012.0012.5013.0013.5014.0014.5015.0015.5016.0016.5017.0017.5018.00 Key challenges: – Reproducibility – Nonspecifically bound glycoproteins on solid supports – Recovery of small glycans from beads – Bead aggregation – Reagent consumption – Incubation times – General robotics issues (e.g. automatic error recovery overnight) Assay Develop- ment Data analysis Robotics

8. IgG GPA Comparison

9. IgG CV Comparison

10. PNGase F time course

11. Minutes 4.004.505.005.506.006.507.007.508.008.509.009.5010.0010.5011.0011.5012.0012.5013.0013.5014.0014.5015.0015.5016.0016.5017.0017.5018.0018.5019.0019.5020.0020.5021.0021.5022.00 18.3 % 19.3 % IGB Robot Serum glycomics program

12. Minutes 4.004.505.005.506.006.507.007.508.008.509.009.5010.0010.5011.0011.5012.0012.5013.0013.5014.0014.5015.0015.5016.0016.5017.0017.5018.0018.5019.0019.5020.0020.5021.0021.50 Reproducibility – Robot (8 replicates)

13. Reproducibility – IGB (8 replicates) Minutes 4.505.005.506.006.507.007.508.008.509.009.5010.0010.5011.0011.5012.0012.5013.0013.5014.0014.5015.0015.5016.0016.5017.0017.5018.0018.5019.0019.5020.0020.5021.0021.5022.00

14. Reproducibility – CVs IGB: 14 peaks > 10% Robot: 7 peaks > 10%, 60% of peaks have better CVs than IGB

15. Comparison of different approaches In-gel block method GlycoBlotGlycoPrep NIBRT platform Time required for the preparation of 96 samples (whole serum glycans) 3 days22 h 6 h (starting from isolated glycoprotein) 14 h Consumables cost per sample 52 EUR62 EUR Throughput96 Sample matrix Serum, plasma, pure glycoprotein Cell culture supernatant, pure glycoprotein Serum, plasma, pure glycoprotein, tissue AutomationpotentiallySweetBlotAssayMap BravoHamilton StarLet Commercial availability Sumitomo BakeliteProzyme/Agilent

16. IgG glycomics workflow – 2AB labelling Anal. Chem. 2013, 85, 8841-8849

17. Comparison of different approaches GlycoBlotGlycoPrep Quick labelling platform (under development) Sample preparation time 22 h 6 h (starting from isolated glycoprotein) < 2 h3 h4 h Consumables cost per sample 52 EUR62 EURcompetitive Throughput96 1-596384 Sample matrix Serum, plasma, pure glycoprotein Cell culture supernatant, pure glycoprotein Serum, affinity purified protein, Skin tissue Serum, affinity- purified protein (80 ug IgG) Affinity- purified protein (15 ug IgG) AutomationSweetBlotAssayMap Bravo NoYes

18. 96-well quick labelling – human IgG

19. 384-well quick labelling – IgM Glycoprofile

20. 384-well quick labelling – IgA Glycoprofile

21. Project Portfolio 2012 Serum glycomics assay development Pancreas cancer study (R. Saldova + E. Kure) 2012-2013 IgG glycomics assay development JIA/UI (with P. Nigrovic) Q1/2013 Glycosylation in animal health - Endometritis study Q2/2013 Glycosylation in animal health - Pregnancy study Q2/2013 Improvement of serum glycomics assay CRC – first large-scale study (GlycoBioM) Q3/2013 Breast cancer study (R. Saldova, V. Haakensen), TB infection study High-throughput assay development + skin glycomics (R. Duke)

22. Application Glycosylation in Uterine Health Uterine disease: ‘Microbial infection and/or pathological inflammation of the uterus’ Humans: Pelvic Inflammatory Disease (PID): Often STIs, causes over 100,000 women to become infertile in the US each year; no single test has adequate sensitivity and specificity to diagnose. Bovine: Metritis/Endometritis.

23. Uterine bacteriology Uterine PathogensPotential pathogensOpportunist Contaminants Escherichia coli Trueperella pyogenes Fusobacterium necrophorum Fusobacterium nucleatum Prevotella spp Acinetobacter spp Bacillus licheniformis Enterococcus faecalis Heamophilus somnus Mannhiemia haemolytics Pastuerella multocida Peptostreprococcus spp Staphylococcus aureus Streptococcus uberis Aerococcus viridans Clostridium butyricum Clostridium perfringens Corynebacterium spp Enterobacte aerogenes Klebsiella pneumoniae Micrococcus spp Providencie rettgeri Providencia stuartii Proteus spp Proprionobacterium granulosa Staphylococcus spp (coag -) A-haemolytic Streptococcus Streptococus acidominimus © R Paralan

24. Uterine disease and its impact on the dairy industry Diagnosis of uterine infection typically occurs after clinical observation of disease between 2 and 5 weeks after calving. Damage to animal health, productivity, and fertility has already occurred. Sought: Reliable early test to accurately diagnose uterine disease in the first few days after calving enable early therapeutic intervention and development of management strategies to reduce the substantial economic and welfare impacts.

25. - Do healthy cows and cows with uterine infection differ in IgG glycosylation? - Can IgG glycosylation be exploited as a biomarker? 98 subjects: clean, endometritis, metritis prepartum ~10 days day 7 pp day 14 pp day 21 pp calving blood sampling and uterine health assessment Health monitoring Ultrasonography Fertility monitoring

26. Peak assignments

27. Representative glycosylation differences healthy endometritis

28. Representative glycosylation differences healthy endometritis

29. Fucosylation Ratios. Day 0 Fucosylation Ratios. Day 7

30. Fucosylation Ratios. Day 14

31. Results from large-scale study

32. Determining predictive power of glycans

33. P-values and AUC Day 14

34. Conclusions Glycans are promising candidates for uterine disease classification Fucosylation is a strong marker Animal health Set-up automated high-throughput assay for fucosylation analysis Assay development Validation of HT quick assay Labelled dextran ladder Future aims

37. GlycoBase Update – Basic Structure NIBRT Collaborations Academic Projects Contract Research Private Clients Data Review Process GlycoBase

38. GlycoBase Update - Overview Unique Structures13991 Individual Measurements11023 Glycans with LC data846 Number of Profiles827 Replicate Sets123 Digest Panels63

39. GlycoBase – New Features New Collections Available Sign-up free application Major Collections Reviewed Digest Panel information In-silico Digest (GlycoDigest) New Searching Functionality Application Programming Interface (API)

40. New Collections CE Data: Heptaglobin IgG Standards RNAse B Transferrin UPLC Milk oligosaccharide: Cow, Dromedary Camel, Goat, Horse, Pig, Sheep Human Serum, IgG HPLC Royle 2008 Paper RP-UPLC Human IgG

41. Graphical Reporting

42. GlycoDigest Available Enzymes: CBG, NVS, XMM, JBH, XMF, ABS, NAN1, BTG, AMF, BKF, JBM, GUH, SPG

44. IgG Structural Assignments GP 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

45. Glycosylation in Animal Health – Small Scale Pilot Study