Authors: Dr Róisín O’Flaherty, Dr Radka Fahey (Saldova) and Prof Pauline Rudd A robotic platform technology developed at the National Institute for Bioprocessing Research and Training (NIBRT) enables high-throughput, automated sample preparation for the diagnosis of cancers, autoimmune diseases and other chronic diseases. The technology allows the release and labelling of N-linked glycans for up to 96 protein or serum samples in less than 15 hours, with the possibility of processing up to 768 samples in tandem (glycans consist of monosaccharide sugars linked together). The research, which was led by Prof Pauline Rudd, was conducted as part of the High Glycan project. The growing need for clinical markers for disease diagnosis, progression and response to therapy is exemplified by the rising demand from patients and health providers worldwide. Global biomarker markets are estimated to grow to $30.6 billion (compound annual growth rate of 16%) in 2020 (1) and glycans are a promising new target. Biomarkers are measurable indicators of a disease. Glycomics is the study of glycan structures in a cell, organism or body fluid. The significance of glycan structures in the onset of disease is widely recognised and changes can be indicative of disease progression and response to therapy, making them suitable targets for biomarkers. High-throughput technologies are necessary to handle large clinical sample sets and, already, the NIBRT platform has contributed to advances in many areas of biomedical research. Complexities in glycan structural analysis have hindered the progress significantly in high-throughput analyses in glycomics, compared to related fields such as proteomics – which is the study of proteins in a cell or organism. The focus of the FP7-funded High Glycan project is to strengthen and develop high-throughput methodologies in this field. [caption id="attachment_19631" align="aligncenter" width="1174"]High Glycan The biological process at the heart of the high throughput platform. IgG1 protein structure reprinted with permission from M.R Wormald and R.A Dwek, Oxford Glycobioloy Institute[/caption]

High-throughput platform

Researchers within NIBRT have developed a robotic platform consisting of a Hamilton Starlet liquid-handling station coupled with high-performance liquid chromatography (HPLC), ultra-performance liquid chromatography (UPLC) and mass spectrometry for glycan analysis of biological matrices including whole serum, plasma and synovial fluid. These platforms have been successfully utilised in glycoprofiling studies in diseases such as cancer, galactosemia, rheumatoid arthritis and diabetes (2-5) and current studies are ongoing in these areas. The automated sample preparation workflows, depending on the type of glycan analysis (whole serum or purified protein), can handle multiple samples simultaneously and has excellent reproducibility. This platform is currently being employed to facilitate biomarker discovery. The platform is versatile and can include glycoprotein purification using affinity chromatography, enzymatic glycan release and fluorescent labelling, followed by quantitative UPLC analysis of released glycans (6) (glycoprotein is a protein with one or more carbohydrate groups attached). Affinity chromatography is a technique used to separate a biochemical mixture, in this case to purify the glycoprotein of interest (such as IgG or IgA) from a complex biological matrix such as serum, which contains proteins, electrolytes, antibodies, salts and other components. The glycans are released from the glycoprotein using enzymatic release, whereby an enzyme (PNGase F) acts like scissors to free the glycans. They are attached to a fluorophore for highly sensitive UPLC detection, i.e. if you imagine that the fluorphore allows the glycans to be illuminated and this can be recorded in the UPLC chromatogram. Glycan structures are assigned to the chromatogram by matching peaks to a database developed at NIBRT (GlycoBase 3.2 ©). Glycobase is a tool that allows computer-assisted preliminary structural assignments. To further elucidate the glycan structures, exoglycosidase digestions can be performed. A series of enzymes can cleave off specific monosaccharides, UPLC analysis can be repeated and this provides characteristic information about the glycans in the sample. Mass spectrometry can further expose glycan structures. Statistical analysis is conducted to assess any characteristic patterns to indicate disease progression.

UntitledCollaborations and robotic platform workflows

Collaborations are ongoing at NIBRT that incorporate the robotic platform workflows:
  • Milk biomarker investigations are conducted for disease in bovine species in collaboration with Prof Mark Crowe at University College Dublin (UCD);
  • Autoimmune disease studies are ongoing in collaboration with Dr Peter Nigrovic at Harvard Medical School;
  • Galactosemia studies are in progress with Prof Eileen Treacy at Trinity College Dublin;
  • Cancer investigation includes prostate cancer studies with Prof William Watson at UCD and Prof Richard O’Kennedy at Dublin City University;
  • Breast-cancer studies with Prof Anne-Lise Børresen-Dale at Oslo University Hospital (OUS, Norway);
  • Pancreatic and colorectal cancer studies with Prof Elin Kure also at OUS; and
  • Gel-free high-throughput glyco-proteomic studies with Prof Janne Lehtio at Karolinska Institutet (Sweden).
The GlycoScience group at NIBRT is a member of the High Glycan consortium, which includes academic and industrial partners across Europe. Together, we are carrying out high-throughput analysis comparison studies of serum and immunologically relevant proteins in disease remission in pregnancy in a rheumatoid arthritis cohort. Author biographies UntitledDr Róisín O’Flaherty is a post-doctoral fellow working in NIBRT on an EU FP7-funded project, High Glycan. Her research interests include carbohydrate biochemistry and robotics with a focus on high-throughput disease diagnosis. She holds a PhD in carbohydrate organic chemistry from Maynooth University, where she designed and synthesised glycolipid immunomodulators for biological evaluation. She was awarded the Endeavour Award Research Fellowship in 2010 and conducted bacterial disease investigations at the University of Melbourne, Bio21. In 2012, she joined Intel Corporation as a process engineer in the area of chemical mechanical processing, where she developed her robotic repertoire. She joined NIBRT in 2015, where she exploits her multidisciplinary background in her current position. Dr Radka Fahey (Saldova) is a starting investigator funded by Science Foundation Ireland on ‘Epigenetic regulation of glycosylation and the impact on chemoresistance in cancer’ from October 2014. She leads a disease research subgroup and her interests include the development and utilisation of high-throughput technologies for N-glycan analysis, disease biomarker discovery in cancer and inflammatory diseases and glycan analysis of various samples. She joined the GlycoScience group in May 2005 as a research assistant and defended her PhD at the Institute of Chemical Technology Prague in November 2007. She continued as a post-doctoral researcher working on cancer glycobiomarkers and glycan analysis and joined the EU FP7-funded project, GlycoHIT, in January 2011. GlycoHIT aims to develop and validate complementary and integrated technologies for glycomic analysis of serum in cancer glycobiomarker discovery, diagnostics and glycotherapeutic monitoring. Prof Pauline Rudd, research professor of glycobiology, heads a research group in NIBRT. She moved to Ireland in 2006 following the decision of the Glycan Sequencing Group at Oxford’s Glycobiology Institute to relocate to Dublin. Prof Rudd was a founding scientist of Wessex Biochemicals (Sigma London), visiting research associate at Scripps Research Institute, CA, visiting professor of biochemistry at Shanghai Medical University PRC, visiting scientist at Ben Gurion University of the Negev, Israel and an Erskine visiting fellow, Canterbury University, New Zealand. She was recently a university reader in glycobiology and senior research fellow in the Glycobiology Institute, Oxford and is currently an honorary professor at St George’s Hospital London and adjunct professor at the Barnett Institute, North Eastern University, Boston. She has a Waters Global Innovation award, an Agilent Thought leader award and an honorary doctorate from the University of Gothenburg. The GlycoScience team has recently been shortlisted for the Royal Irish Academy of Science/American Chamber of Commerce award 2015. References 1. D. Shields, R. Deshmukh (Allied Market Research, 2013). 2. B. Adamczyk, T. Tharmalingam, P. M. Rudd, Biochim Biophys Acta 1820, 1347 (Sep, 2012). 3. K. P. Coss et al., J Proteome Res 13, 385 (Feb 7, 2014). 4. S. Albrecht, L. Unwin, M. Muniyappa, P. M. Rudd, Cancer Biomark 14, 17 (Jan 1, 2014). 5. G. Thanabalasingham et al., Diabetes 62, 1329 (Apr, 2013). 6. H. Stockmann, B. Adamczyk, J. Hayes, P. M. Rudd, Anal Chem 85, 8841 (Sep 17, 2013).