Biopolymers: Analysis of oligosaccharides, proteins and peptides and their posttranslational modifications

Electromigrative separation techniques are ideally suited for the analysis of proteins and peptides which can all be sufficiently charged at pH values accessible for CE. This enables e.g. the simultaneous analysis of all analytes in a single analytical run at pH~2. In contrast, both chromatographic techniques commonly used for protein and peptide analysis (RP-HPLC and HILIC) separate analytes based on polarity differences, which may become critical for analytes of extreme polarity. We use various modes of electromigrative separation techniques such as capillary electrophoresis, capillary isoelectric focusing and size sieving electrophoresis (SDS-CE).

The coupling of isoelectric focussin with mass spectrometry is very difficult due to the high ampholyte concentration. Our idea is to identify IEF separated proteins via CE-MS in a second dimension to IEF in multidimensional format.

However, capillary electrophoresis may suffer from analyte adsorption onto the inner capillary wall. We circumvent adsorption phenomena via suitable capillary coating strategies. With them, we were able to successfully analyze critical peptides related to diagnosis or therapy of Alzheimer’s disease with extreme physicochemical properties: these peptides show either anextreme pI-value (above 11) or are extremely hydrophobic which renders thei chromatographic analysis very challenging. With our capillary coatings, both analyte classes could be separated using the same analytical method.

For method optimization, we also investigate solutions for the optimization and development of new capillary coatings, which, in addition to analyte suppression, allow the tailored adjustment of electroosmotic flow direction and velocity. Analyte detection is usually performed via high resolution mass spectrometry, but also conventional UV detection or sensitive fluorescence detection.

One of the most important posttranslational modifications is protein glycosylation, which is highly heterogeneous due to its complex biosynthesis. With electromigrative separation techniques the separation of glycans, glycopeptides but also intact glycoproteins is possible. Very elegantly, the characterization of protein deamidation for pharmaceutical samples is possible. For biomarker discovery, we here also address the large range of protein concentrations in plasma using ligand libraries. Regarding endogenous or pharmaceutical peptides, electromigrative separation techniques can aid in case of extremely hydrophobic or hydrophilic/highly charged analytes.

Further aspects of our work include sample preparation strategies in order to reduce the enormous concentration range of protein in human blood.

Cooperations:

• Katalin Barkovits, Katrin Marcus, Medizinisches Proteom Center, Ruhr-Universität Bochum

Publications:

1. CE-MS for the analysis of amyloid beta peptides as biomarkers for Alzheimer’s disease
   K. Barkovits, M. Pattky, J. Wiltfang, C. Huhn, K. Marcus; submitted 2015
2. Strategies for statically adsorbed coatings for high separation efficiency and resolution in CE-MS peptide analysis
   M. Pattky, K. Barkovits, O. Weiergräber, C. Huhn, Meth. Mol. Biol. 2015, submitted
3. Influence of the electroosmotic flow velocity on the separation performance in CE-MS peptide analysis using different capillary coatings: a comparative study
   M. Pattky, C. Huhn, Anal. Bioanal. Chem. 2013, 405, 225-237
4. Synthesis of nucleotide-activated disaccharides with recombinant β3-galactosidase C from Bacillus circulans
   C. Kamerke, M. Pattky, C. Huhn, L. Elling, J. Mol. Catal. B: Enzymatic 2013, 89, 73-81
5. Synthesis of UDP-activated oligosaccharides with commercial β-galactosidase from Bacillus circulans under microwave irradiation
   C. Kamerke, M. Pattky, C. Huhn, L. Elling; J. Mol. Catal. B: Enzymatic 2012, 79, 27-34
6. Robust and high-throughput sample preparation for (semi-)quantitative analysis of N-glycosylation profiles from plasma samples
   L. R. Ruhaak, C. Huhn, C. A. M. Koeleman, A. M. Deelder, M. Wuhrer, Methods Mol. Biol. – Quantitative Methods in Proteomics 2012 , 893, 371-385
7. Hexapeptide library as a universal tool for sample preparation in protein glycosylation analysis
   C. Huhn, L. R. Ruhaak, M. Wuhrer, A. M. Deelder, J. Proteomics 2012, 75, 1515-28
8. Protein glycosylation analysis with capillary-based electromigrative separation techniques
   M. Pattky, C. Huhn, Bioanal. Rev. 2010, 2, 115-155
9. Optimized workflow for preparation of APTS-labeled N-glycans allowing high-throughput analysis of human plasma glycomes using 48-channel multiplexed CGE-LIF
   L. R. Ruhaak, R. Hennig, C. Huhn, M. Borowiak, R. J. Dolhain, A. M. Deelder, E. Rapp, M. Wuhrer, J. Proteome Res. 2010, 9, 6655-6664
10. Glycan labeling strategies and their use in identification and quantification
    L. R. Ruhaak, G. Zauner, C. Huhn, C. Bruggink, A. M. Deelder, M. Wuhrer, Anal. Bioanal. Chem. 2010, 397, 3457-3481
11. IgG glycosylation analysis
    C. Huhn, M. Selman, R. Ruhaak, A. M. Deelder, M. Wuhrer, Proteomics 2009, 9, 882-913
12. A HILIC-based high-throughput sample preparation method for N-glycan analysis from total human plasma glycoproteins
    L. R. Ruhaak, C. Huhn, W.-J. Waterreus, A. R. de Boer, C. Neusüß, C. H. Hokke, A. M. Deelder, M. Wuhrer, Anal. Chem. 2008, 80, 6119-6126
13. Das Süße im EPO - Glykoproteincharakterisierung mit Kapillarelektrophorese-Massenspektrometrie
    C. Neusüß, S. Vaas, C. Huhn, GIT Laborfachzeitschrift 2008, 1, 22-24