ESR12: Glycans, glycolipids and gangliosides related to dopaminergic cell death?
Csaba Váradi (NIBRT – The National Institute for Bioprocessing Research & Training)
Supervisor: Jonathan Bones
Csaba's research is focused on the development of high sensitivity capillary electrophoresis (CE) based methods with either laser induced fluorescence detection (LIF) or mass spectrometry (MS) for the quantitative determination of alterations in protein N-glycosylation. Developed methods will then be applied to determine changes in the serum N-glycome in patients diagnosed with Parkinson's disease. Additionally, the developed CE-LIF and CE-MS technology will then be transformed to facilitate the analysis of glycolipid head groups using in vitro neuronal models. Once available, these methods will then be used to study alterations in glycosylation at both the glycoprotein and glycolipid level using the in vitro system which has undergone modification at Trinity College Dublin using advanced molecular biology methods for receptor manipulation.
The specific aims of the project are:
- To characterize serum and cerebrospinal fluid N-glycans and reveal their biomarker potential in Parkinson's and Alzheimer's disease using the developed methods
- To reveal the possible correlations between glycosylation changes and disease progression in Parkinson's and Alzheimer's disease using in vitro neuronal models.
• Highly sensitive capillary electrophoresis (CE) based methods were developed, with either laser-induced fluorescence (LIF) or mass spectrometry (MS) based detection for the quantitative analysis of the serum and CSF based N-glycomes using commercially sourced serum and cerebrospinal fluid. An advanced separation system was also developed, which provides higher resolution than current commercially available solutions for glycan separation by CE and enables facilitated transfer between CE instrumentation, i.e. Beckman PA800 Plus CE-LIF and Agilent 7100-6520 CE-MS.
• Following development and optimization of the separation conditions, a glycan analysis was performed following derivatisation of the enzymatically released oligosaccharides using APTS or 2-aminobenzoic acid to impart electrophoretic mobility and facilitate high sensitivity detection. A quantitative glycomic method employing sample derivatisation using 12C6/ 13C6 2-aminobenzoic acid for twoplex based quantitation was developed. The performance of the method has been evaluated using monoclonal antibody glycans and a resulting paper is in preparation for submission for publication.
• An in-house cell bank has been created, which has generated sufficient cellular material to commence glycomic profiling of the cells.
(i) Peer reviewed publications and monographs:
• Quantitative Twoplex Glycan Analysis Using 12C6 and 13C6 Stable Isotope 2-Aminobenzoic Acid Labelling and Capillary Electrophoresis Mass Spectrometry
[submitted to Analytical and Bioanalytical Chemistry]
(ii) Abstracts to meetings A poster has been presented on The "Opportunities in Biopharma Research" workshop on Tuesday 29th September 2015.
(i) Local level
- Presentations and professional communications course at NIBRT in October 2014 delivered by Vanessa Cole from Training Works.
- Mammalian cell culture training.
- Instrument specific training on Beckman PA800 Plus CE-LIF and Agilent Technologies 7100- 6520 CE-MS.
- Data analysis and processing using Agilent Technologies Mass Hunter software platforms and GlycoWorkBench and other glycobioinformatics tools.
- NIBRT training afternoon on biopharma processing in July 2015.
(ii) Network level
- Neuronal Cell Metabolism
- Training in Mitochondrial and cellular respiratory physiology
- NMR Mini Boot Camp of BioBank Analyses and Metabolomic Transformation
- Fluorescence and electron microscopy imaging of cells