Completed Projects

The open-source python software suite glyXtool^MS facilitates the semi-automated analysis of N- and O-glycopeptide mass spectrometry fragmentation data. The software adds new ana­lysis tools for the pipeline engine of OpenMS, which was developed for proteomics experiments. These include a filter to discriminate between glycopeptide and peptide spectra, glycopeptide identification via precursor matching to glycan compositions and in-silico generated peptide sequences, fragment ion annotation as well as scoring tools. The analysis results can be visualized within the glyXtool^MS Evaluator, which also enables further manual analysis, inspection and verification. glyXtool^MS is freely available online on https://github.com/glyXera/glyXtoolMS licensed under the GPL-3.0 open-source license. [more]

For site-specific glycosylation analyses, glycopeptide-based glycoproteomics using mass spectrometry is the method of choice. The aim of such analyses is the identification of occupied glycosylation sites, including the quantification of the site occupancy (macroheterogeneity), as well as identification, structural characterization and quantification of all glycans attached to the individual glycosylation sites (microheterogeneity). Over the last years we have developed and established mass-spectrometry-based methods and workflows that enable the site-specific and in-depth analysis of N- and O-glycopeptides. [more]

The analysis of protein glycosylation is of increasing importance in glycobiological research as well as for monitoring and quality control in development and production of biopharmaceuticals. In order to perform better and faster during the discovery phase, R&D, (clinical) trials, and approval of recombinant proteins and vaccines, automated high-throughput (HT) and high-resolution glycoanalytics is mandatory. [more]

Most current methods to detect glycans and resolve their structures require enzymatic treatment coupled to analytical techniques. Enzymes termed endoglycosidases that release intact glycans from glycoconjugates are used at the front end of workflows that use “free” glycans as starting material. Highly specific exoglycosidases are often used to sequentially remove sugar units one by one to enable elucidation of a glycan’s composition. Some glycans can be modified with additional chemical groups (e.g., sulfate, phosphate etc) in specific positions, often called post-glycosylation modifications (PGMs).  [more]

Glycosylation is one of the most common, most complex, and most important posttranslational modifications of proteins. A large proportion of the proteome in eukaryotic systems is glycosylated. This highly diverse modification is involved in many cellular functions, including modulation and mediation of signaling events, cell-cell and cell-matrix interaction, and extracellular as well as intracellular traffic. Glycosylation provides a variety of functions, like the control of protein folding, the regulation of the immune response, and the modulation of physical, chemical, and biological properties of proteins of the blood circulation. Also many diseases like diabetes, cancer, rheumatoid arthritis, cardiovascular diseases, Alzheimer's disease, and Creutzfeld-Jakob disease are associated with changes in the glycosylation of proteins. To further increase knowledge and understanding in glycobiology the comprehensive investigation of the entire complement of sugars (glycome), large-scale clinical- and population-based studies, focused on biomarker discovery are needed. [more]

Multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) is a powerful analytical technique that allows the sensitive, selective, robust, and high-throughput identification and quantification of released N-glycans and free oligosaccharides (glycoprofiling). Due to the use of fluorescent labels and the capillary-based gel electrophoretic separation minute amounts of analytes – including structural isomers – can be separated by size, charge and shape with high peak capacity and detected with high sensitivity, without encountering typical caveats of chromatography-based separation techniques, such as selectivity, carry-over, or overloading effects.  [more]

In cooperation with the glyXera GmbH in Magdeburg/Germany, a method for HT/HP/HR glycoanalysis, based on multiplexed capillary gel electrophoresis with laser induced fluorescence detection (xCGE-LIF) utilizing a DNA-sequencer, is developed at the MPI. The application of this technique to glycoanalysis using instruments with up to 96 capillaries in parallel, results in massive reduction of the effective separation time per sample combined with an impressive sensitivity achieved due to LIF detection and therefore, shows high potential for HT/HP/HR glycoprofiling of glycoconjugates. [more]

The analysis of protein glycosylation became of increasing importance in glycobiological research, as well as for bioprocess monitoring during development and production of biopharmaceuticals and vaccines in the recent past. In order to enhance and improve the existing glycoanalytical toolbox, automated high-throughput, high-reso­lu­tion and highly sensitive but robust analysis methods with automated data processing and analysis are required.  Multiplexed capillary gel electrophoresis with laser induced fluorescence de­tection (xCGE-LIF), a quite feasible electrokinetic separation technique using stand­ard DNA sequencer equipment, has been developed for glycoprofiling of fluorescently labeled gly­cans. However, when it comes to data analysis bioinformatics software is still lacking for proc­ess­ing experimental data. [more]

Metaproteomics represents the large scale characterization of the entire protein complement expressed by a microbial community at a given time point and under defined environmental conditions. This research area aims at assessing the immediate catalytic potential of microbiota. However, the investigation of complex microbial communities by proteomic methods remains a challenging task as in contrast to pure-culture proteomics, metaproteome samples are heterogeneous and much more complex.  [more]

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has become a standard method in the field of proteomics by combining fast and reproducible measurements with high mass accuracy. Peptides can be analyzed with high confidence, using standard MALDI-matrices like 2,5-dihydroxybenzoic acid (DHB), and α-cyano-4-hydroxycinnamic acid (CHCA). An upcoming field of application for MALDI-TOF-MS is the analysis of glycoconjugates, like glycans and glycopeptides. Compared to peptides, those glycoconjugates have different requirements on the MALDI-matrix that is used. Here the development of new MALDI-matrices can greatly improve the detection and quantification of glycans and glycopeptides. [more]

The analysis of biomolecules by mass spectrometry (MS) put rather severe constraints on the ionization method, as far as its ability to produce intact biomolecular ions (without uncontrollable fragmentation) is concerned. With the advent of the electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), two ionization techniques of unprecedented softness appeared. Due to the increasing need for powerful analytics of complex mixtures, coupling of separation methods, such as high-performance liquid chromatography (HPLC), with the mass spectrometric detection appeared prerequisite for dealing with complex mixtures of biomolecules, such as protein digests of cell lysates. Concomitantly, the quest for the best LC-MS interface started early in the 1970's. [more]

Another project utilizing this glycoanalysis approach is the HT characterization of human milk oligosaccharides (HMOS) with respect to the improvement of artificial, respectively, functional food. The project is realized in cooperation with Nutricia Research/Danone, Human Milk Research in Utrecht/Netherlands. The HMOS fraction primarily consists of lactose and a large variety of neutral and acidic oligosaccharides. The characterization of HMOS is essential to understand the relationship between structure and biological effect. However, as the role and significance of HMOS is still not fully understood, identification and characterization of oligosaccharide structures is required. [more]

In cooperation with Prof. Scharfenberg at the Applied University Emden/Leer in Emden/Germany an adherent Madin-Darby canine kidney (MDCK) cell was adapted to growth in suspension. In mammalian cell culture growth in suspension and in chemically defined medium is desirable for many production processes. However, for vaccine production processes a switch to growth in suspension is often challenging as many viruses seem to depend on cell lines difficult to adapt. Furthermore, cell-specific productivity is often reduced after adaptation. [more]

Glycosylation is considered as a critical quality attribute for the production of biopharmaceuticals, i.e. recombinant proteins such as monoclonal antibodies. In recent years, glycosylation of antigens is also considered for the manufacturing of viral vaccines. As yet, however, glycosylation and immunogenicity of only few viral antigens was characterized in the context of host cell selection, cultivation parameters, processes mode (batch, fed-batch, perfusion), and purification. Using xCGE-LIF based glycoanalysis  [more]