Method Development for Multiplexing CGE-LIF Based Glycoanalysis

Motivation

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 was 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. Therefore, the method shows high potential for HT/HP/HR glycoprofiling of glycoconjugates.

Aim of the project

The aim of this project was to further investigate and to improve this innovative approach for use in various fields of application. Therefore, the sample preparation method and workflow were further optimized with respect to performance and feasibility regarding HT. In addition, separation parameters (e.g.: gel matrix composition, voltage or capillary length) were evaluated regarding HP and HR to find the optimum for an automated separation for each of a set of different types of samples.

 

Publications

Hennig, R.; Rapp, E.; Kottler, R.; Cajic, S.; Reichl, U.: N -Glycosylation Fingerprinting of Viral Glycoproteins by xCGE-LIF. In: Carbohydrate-Based Vaccines: Methods and Protocols, pp. 123 - 143. Springer, New York (2015)
Borowiak, M.; Hennig, R.; Kottler, R.; Reichl, U.; Rapp, E.: Tailored methods for CGE-LIF based multiplexed high-throughput glycoanalysis with respect to sample characteristics. In Glycoconjugate Journal, 28 (5 ), p. 332. 21st International Symposium on Glycoconjugates - Glyco 21, Wien, Östereich, August 21, 2011 - August 26, 2011. (2011)
Schwarzer, J.; Rapp, E.; Reichl, U.: N-Glycan Analysis by CGE-LIF – Profiling Influenza A Virus Hemagglutinin N-Glycosylation during Vaccine Production. Electrophoresis 29 (20), pp. 4203 - 4214 (2008)

 

Patents

[1] E Rapp, J Schwarzer, U Reichl, C Bohne: Method for automated high throughput identification of carbohydrates and carbohydrate mixture composition patterns as well as systems therefore. European patent: EP2533039 (A1) (2008).

[2] E Rapp, J Schwarzer, U Reichl, C Bohne: Method for automated high throughput identification of carbohydrates and carbohydrate mixture composition patterns as well as systems therefore. US patent: US2013075258 (A1) (2009).

Software Development for LC-MS/MS Based Metaproteomics

Motivation

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 aimed 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 have a much higher complexity. Another problem constitutes the fact that only a small proportion of the microbial genome is sequenced, which leads to insufficient MS-based protein identification results. As a result a large quantity of fragmentation mass spectra remains unidentified due to the lack of appropriate protein sequence databases. Another important aspect is the functional analysis of complex microbial communities, as it constitutes an essential prerequisite for scientific endeavors such as improving the eco­nomic efficiency of biogas or waste water treatment plants, environ­ment­al remediation or investigating the complex microbial interactions in the human gut. 

Aim of the project

Mainly due to the lack of appropriate software solutions, the analysis and interpretation of data derived from LC-MS/MS experiments presented a major bottleneck in metaproteomics. In order to tackle the aforementioned challenges and overcome the limits of existing software solutions an improvement and adaptation of protein identification algorithms and database infrastructure was required. For that purpose, the software tool MetaProteomeAnalyzer (MPA) was developed, a client-server application tailored for the in-depth analysis of metaproteomics data that allows for the automated functional and taxonomic characterization of contained proteins in the samples.

Publications

Muth, T.; Kohrs, F.; Heyer, R.; Benndorf, D.; Rapp, E.; Reichl, U.; Martens, L.; Renard, B. Y.: MPA Portable: A Stand-Alone Software Package for Analyzing Metaproteome Samples on the Go. Analytical Chemistry 90, pp. 685 - 689 (2018)
Muth, T.; Behne, A.; Heyer, R.; Kohrs, F.; Benndorf, D.; Hoffmann, M.; Lehteva, M.; Reichl, U.; Martens, L.; Rapp, E.: The MetaProteomeAnalyzer: A Powerful Open-Source Software Suite for Metaproteomics Data Analysis and Interpretation. Journal of Proteome Research 14 (3), pp. 1557 - 1565 (2015)
Muth, T.; Kolmeder, C. A.; Salojärvi, J.; Keskitalo, S.; Varjosalo, M.; Verdam, F. J.; Rensen, S. S.; Reichl, U.; de Vos, W. M.; Rapp, E. et al.; Martens, L.: Navigating through metaproteomics data: A logbook of database searching. Proteomics 15 (20), pp. 3439 - 3453 (2015)
Muth, T.; Benndorf, D.; Reichl, U.; Rapp, E.; Martens, L.: Searching for a needle in a stack of needles: challenges in metaproteomics data analysis. Molecular BioSystems 9 (4), pp. 578 - 585 (2013)
Hanreich, A.; Schimpf, U.; Zakrzewski, M.; Schlüter, A.; Benndorf, D.; Heyer, R.; Rapp, E.; Puhler, A.; Reichl, U.; Klocke, M.: Metagenome and metaproteome analyses of microbial communities in mesophilic biogas-producing anaerobic batch fermentations indicate concerted plant carbohydrate degradation. Systematic and Applied Microbiology 36 (5), pp. 330 - 338 (2013)
Heyer, R.; Kohrs, F.; Benndorf, D.; Rapp, E.; Kausmann, R.; Heiermann, M.; Klocke, M.; Reichl, U.: Metaproteome analysis of the microbial communities in agricultural biogas plants. New biotechnology 30 (6), pp. 614 - 622 (2013)

Elucidation of the Carbohydrate Composition of Human Milk

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 was 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.

Multiplexed capillary gelelectrophoresis with laser induced fluorescence detection (xCGE-LIF), allowed the generation of HMOS "fingerprints" (normalized electropherograms). In combination with an in-house HMOS database, the system was used for in-depth glycoanalysis, i.e.: structural elucidation of each single compound and its relative quantification. The method developed was tested on various human milk samples (time series of different donors). Several factors were identified to be responsible for differences in HMOS-composition. These included the origin of the mother, the lactation time point, and genetic determinants, e.g.: the secretor-gene (Se) and the Lewis blood group system with the Lewis gene (Le).

Overall, the xCGE-LIF based HT glycoanalysis system enabled qualitative and quantitative glycoanalysis of large donor cohorts, and of cohort time series along the course of lactation.

Publications

Kottler, R.; Hennig, R.; Mank, M.; Stahl, B.; Reichl, U.; Rapp, E.: High-Throughput Characterization of the Human Milk Oligosaccharide Composition Utilizing a Multiplexing Capillary DNA Sequencer. In Glycoconjugate Journal, 28 (5 - Special Issue : XXI International Symposium on Glycoconjugates), p. 330. 21st International Symposium on Glycoconjugates (Glyco 21), Wien, Östereich, August 21, 2011 - August 26, 2011. (2011)
Kottler, R.; Mank, M.; Hennig, R.; Müller-Werner, B.; Stahl, B.; Reichl, U.; Rapp, E.: Development of a high-throughput glycoanalysis method for the characterization of oligosaccharides in human milk utilizing multiplexed capillary gel electrophoresis with laser-induced fluorescence detection. Electrophoresis 34 (16), pp. 2323 - 2336 (2013)

Proteomics Along the Adaptation of an Adherent MDCK Cell Line to Growth in Suspension

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 animal 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.

In this project, adherent growth versus growth in suspension for a MDCK cell line was investigated on the proteome level. Interestingly, significant changes on the protein level during adaptation of an adherent MDCK cell to growth in suspension could be identified. Differences in proteomic profile were detected during cell line adaptation as well as during exponential and stationary growth of the adherent and the suspension cell line. Most of the differentially expressed proteins were subsequently identified. The findings gave new insights into key characteristics of cell line adaptation to growth in suspension.

 

Publications

Kluge, S.; Benndorf, D.; Genzel, Y.; Scharfenberg, K.; Rapp, E.; Reichl, U.: Monitoring changes in proteome during stepwise adaptation of a MDCK cell line from adherence to growth in suspension. Vaccine 33 (35), pp. 4269 - 4280 (2015)

Analysis of Virus-Host Cell Interaction on the Proteome Level

Motivation

This project was related to other genomic and metabolic approaches, all focusing on the dynamic interaction of viruses with their mammalian host cells during vaccine production. Therefore, changes in the expression of host cell proteins in various mammalian cells after infection with different virus strains were characterized. In addition, proteome changes related to cultivation conditions were investigated.

Aim of the project

Using two dimensional differential gel electrophoresis and mass spectrometry (Figure 1) we identified differentially expressed host cell proteins over the course of an infection phase. Different cell lines, like Vero and MDCK cells, are hosts for cell-based vaccine production. The results were correlated with integrated process data and gave detailed insight into vaccine production on the cellular level. This knowledge contributed to a better characterization und understanding of the complexity of virus-host cell interactions.

 

Publications

Vester, D.; Rapp, E.; Gade, D.; Genzel, Y.; Reichl, U.: Quantitative analysis of cellular proteome alterations in human influenza A virus-infected mammalian cell lines. Proteomics 9 (12), pp. 3316 - 3327 (2009)
Vester, D.; Rapp, E.; Kluge, S.; Genzel, Y.; Reichl, U.: Virus-host cell interactions in vaccine production cell lines infected with different human influenza A virus variants: A proteomic approach. Journal of Proteomics 73 (9), pp. 1656 - 1669 (2010)
Kluge, S.; Rourou, S.; Vester, D.; Majoul, S.; Benndorf, D.; Genzel, Y.; Rapp, E.; Kallel, H.; Reichl, U.: Proteome analysis of virus-host cell interaction: rabies virus replication in Vero cells in two different media. Applied Microbiology and Biotechnology 97 (12), pp. 5493 - 5506 (2013)
Kluge, S.; Genzel, Y.; Laus, K.; Serve, A.; Pflugmacher, A.; Peschel, B.; Rapp, E.; Reichl, U.: Ezrin and HNRNP expression correlate with increased virus release rate and early onset of virus-induced apoptosis of MDCK suspension cells. Biotechnology Journal 11 (10), pp. 1332 - 1342 (2016)
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