Importance of sample clean-up for 2AB labelling No clean-up
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
Reproducibility – CVs IGB: 14 peaks > 10% Robot: 7 peaks > 10%, 60% of peaks have better CVs than IGB
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
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
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)
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.
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.
- 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
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
GlycoBase Update – Basic Structure NIBRT Collaborations Academic Projects Contract Research Private Clients Data Review Process GlycoBase
GlycoBase Update - Overview Unique Structures13991 Individual Measurements11023 Glycans with LC data846 Number of Profiles827 Replicate Sets123 Digest Panels63
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)
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