The IPB has once again been recognized for its exemplary actions in terms of equal opportunity-oriented personnel and organizational policies and has received the TOTAL E-QUALITY certification for the…
The Plant Science Student Conference (PSSC) has been organised by students from the two Leibniz institutes, IPK and IPB, every year for the last 20 years. In this interview, Christina Wäsch (IPK) and…
Weissenborn, M. J.; Debecker, D. P.; Golten, S.; Linclau, B.; Turner, N. J.; Flitsch, S. L.;Development of a Solid Phase Array Assay for the Screening of Galactose Oxidase Activity and for Fast Identification of InhibitorsProtein Peptide Lett.24742-746(2017)DOI: 10.2174/0929866524666170724114348
Background: Galactose oxidase (GOase) catalyses the highly selective oxidation of terminal galactosides on a wide range of natural glycoconjugates and has found wide applications in biotechnology – particularly in biocatalysis. GOase is copper dependent and uses oxygen to oxidise the C6-primary alcohol of galactose and produces hydrogen peroxide. The enzyme activity can be conveniently assessed by a colorimetric assay.Objectives: The objective of the present study was to develop an assay system, which is independent of the hydrogen peroxide formation to identify possible fluorinated GOase inhibitors. In case that the inhibitor bears a primary or secondary alcohol, it could also be oxidised by the enzyme. In such case, the colorimetric assay is not able to distinguish between substrate and inhibitor, since oxidation of both molecules would result in the formation of hydrogen peroxide.Methods: D-galactose (D-Gal) was immobilised onto a gold surface functionalised by selfassembled monolayers (SAMs,). A GOase solution was then added to the surface in a droplet for a certain period of time and thereafter washed away. The activity of GOase on the immobilised D-Gal can then be quantified by MALDI-ToF MS.Results: For inhibition studies, GOase was incubated together with 62.5 mM of deoxy-fluorinated monosaccharides on the D-Gal displaying platform. Five deoxy-fluorinated D-Gal showed a >50% inhibition of its activity. The array system has been moreover utilised to determine the apparent IC50 value of 3-F-Gal 15 as a proof of principle.Conclusion: The developed array platform allows the fast identification of GOase substrates and inhibitors from a library of deoxy-fluorinated sugars using MALDI-ToF MS as a label–free readout method. In addition, the enzymatic reaction enables for the in situ activation of sugar-coated surfaces to bioorthogonal aldehydes, which can be utilised for subsequent chemical modifications.
Publications
Spiller, S.; Frolov, A.; Hoffmann, R.;Quantification of Specific Glycation Sites in Human Serum Albumin as Prospective Type 2 Diabetes Mellitus BiomarkersProtein Peptide Lett.24887-896(2017)DOI: 10.2174/0929866524666170202124120
Background: Type 2 diabetes mellitus (T2DM) is the most common lifestyle disease affecting all countries. Due to its asymptomatic onset, it is often diagnosed after irreversible vascular complications have been initiated. Therefore, specific markers characteristic for very early disease stages and suitable for early diagnostics are required. Glycation of plasma proteins, such as human serum albumin (HSA), has been often suggested as marker. However, the total glycation degree of HSA does not provide sufficient information about short-term fluctuations of blood glucose concentrations due to the large number of glycation sites. Analysis of individual modification sites might be more informative, but methods for reliable quantifications are still missing.Objective: The main objective of this study was to establish and qualify a method of analysis applicable to sensitive and precise quantification of glycations sites in plasma proteins.Methods: Plasma samples obtained from diabetic patients and non-diseased individuals were separated from low-molecular weight compounds, digested with trypsin, enriched for glycated peptides by boronic acid affinity chromatography (BAC), desalted by solid phase extraction (SPE), and separated by RP-HPLC coupled online to ESI-QqQ-MS. Quantification relied on multiple reaction monitoring (MRM) of multiple glycation sites identified in plasma proteins using a stable isotope dilution approach or internal standardization.Results: The data presented here suggests high selectivity and precision (relative standard deviations below 10%) of the overall approach appearing to be well suited for the identification of prospective biomarkers. Six glycated peptides corresponding to different glycation sites of HSA were present in plasma samples obtained from T2DM patients at significantly higher levels than in non-diabetic men matched for age. Additionally, each of the studied glycation site of HSA appeared to be affected at different degrees.Conclusion: The presented approach enables the sensitive and robust quantification of prospective T2D biomarkers promising for clinical diagnostics.