The Institute of Chemistry at the Slovak Academy of Sciences offers expertise in analysing the structure, conformation, and dynamics of mono-, oligo- and poly-saccharides, their derivatives and glycoconjugates, as well as in weak intermolecular interactions between proteins and carbohydrate molecules. Available technology includes Physico-analytical methods such as high-resolution nuclear magnetic resonance (NMR), mass spectrometry (MS), infrared (IR) and Raman spectroscopy, surface plasmon resonance (SPR), atomic force microscopy (AFM), differential scanning calorimetry (DSC), and RTG diffractometry.
The NMR laboratory can offer all high-resolution methods (1D, 2D) that are necessary for structural analysis of glycans.
The institute can also offer analysis of protein glycosylation by mass spectrometry, including determination of the intact glycoprotein mass, N-glycan profiling and mapping protein N-glycosylation sites. The outcome of this service is the list of N-glycans, their composition/structure based on MS and MS/MS data and a list of potential glycosylation sites where the glycans covalently attach to the protein of interest.
Glycosylation is an essential, non-template, post-translational modification of proteins that affects their properties, function, folding, activity and interactions. Comprehensive knowledge of glycosylation is crucial for biopharmacological products (e.g. therapeutic antibodies and enzymes) or other recombinant proteins expressed by hosts different from the protein’s primary origin.
Glycobiology has not yet been offered through the Instruct-ERIC service catalogue but the analyses of the saccharide structures and their dynamics will complement and expand already established methods in the infrastructure (protein or nucleic acids structure analyses). Apart from the experimental methods (NMR, MS, IR, Raman,…) the Institute provides expertise in the theoretical analysis of carbohydrate structures by means of quantum-chemical approaches, such as density functional methods (DFT).
Samples can be sent for analysis by post or courier. Glycosylation is a stable modification and even in case of protein degradation, the corresponding glycoprofile can be determined. Depending on the analysis required, the proteins can be delivered at room temperature as lyophilised or precipitated samples, or on dry/wet ice when in solution. The results will be reported and discussed online, meaning no personal travel is required.
• Intact glycoprotein mass will be determined by MALDI TOF analysis using ultrafleXtreme MALDI TOF/TOF instrument (Bruker). The level of glycosylation can be estimated by comparison of the intact glycoprotein with its deglycosylated form.
• Analysis of N-glycoprofile includes protein denaturation, deglycosylation, isolation of released glycans with subsequent permethylation and analysis by MALDI TOF/TOF mass spectrometry (ultrafleXtreme, Bruker).
• The protein glycosylation sites will be determined by proteolytic cleavage, heavy atom incorporation upon hydrolase treatment and proteomic analysis of the peptide mixture using nanoHPLC-Orbitrap MS/MS (ThermoScientific). Quantitative proteomic analysis will provide an estimate of the glycan occupancy of the individual sites.
• Primary and secondary structure of oligo- and polysaccharides by applying 1D and 2D high-resolution NMR. 3D structural analysis can also be performed combining NMR spectroscopy and theoretical analysis (mainly DFT, but other methods are also available).
N-glycosylation profiles of both single isolated (e.g. recombinant) proteins and complex protein mixtures, such as membrane fraction of cell lysate, human serum, etc. can be determined at the Institute of Chemistry.
For glycomic and glycoproteomic analyses, native as well as denatured proteins are suitable. Prior to the analysis, reliable determination of protein concentration in the sample is essential.
Around 20/100 ug of isolated protein/protein mixture is usually required for glycoproteomic and 100/500 ug of protein/protein mixture for glycomic analysis.
The call will have two openings, we advise that applicants apply as soon as possible. The first approvals will be made on 30 August, and the second on 30 September.
Samples for laboratory analysis must be received within one month of award, and all analysis will be completed by 30 November.
Researchers who are based in Instruct-ERIC and Instruct-ULTRA participating countries are eligible to apply: Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Israel, Italy, Latvia, Lithuania, Netherlands, Portugal, Slovakia, Spain, Sweden, UK, as well as all EMBL researchers.
Applicants can be pre-doctoral scientists (PhD students), or postdoctoral researchers from an academia or industry (non-proprietary research).
How to apply:
To apply for the Open Call, you will first need to set up a user account on ARIA. When you click to submit a new proposal for this call, you will be automatically directed to a page where you can set up or log in to an ARIA account.
Applications should focus on the benefit to the applicant’s research. All proposals will be peer reviewed, and awards made based on the quality of science. Funding will be available to cover instrument access.
Within 3 months of the completion of the internship platform access, applicants must give visit feedback and submit a short report about their research experience.
All proposals submitted by industry will be kept confidential. Any questions about legal agreements can be addressed to the host research centre.