ausblenden:
Schlagwörter:
-
Zusammenfassung:
The analysis of protein glycosylation became of increasing importance in research, as well as for monitoring production processes of biotechnologically produced pharmaceuticals, in the recent past. Applications in both fields require high-throughput (HTP) and high-resolution (HR) analytical methods that allow fast and robust profiling of protein glycosylation on large sample sets. To characterize these complex posttranslational modifications of proteins several analysis methods were developed in the last three decades. Besides several mass spectrometry and liquid chromatography based analysis techniques, electromigrative separation techniques became apparent during the recent past. Especially capillary gel-electrophoresis with laser-induced-fluorescence-detection (CGE-LIF), using standard DNA sequencing equipment, has been developed for HTP glycoprofiling of APTS-labeled glycans [1]. The application of this technique with up to 96 capillaries in parallel, results in massive reduction of the effective separation time per sample combined with an impressive sensitivity due to LIF detection. Here we present an optimized sample preparation procedure for N-glycan-profiling by CGE-LIF, enabling the HTP glycosylation analysis of e.g. human plasma N-glycome [2]. After enzymatic glycan-release, N-glycan labeling with APTS was performed, using 2-picoline borane as a non-toxic reducing agent. Reaction conditions were optimized for a high labeling efficiency, short handling times and only limited loss of sialic acids. A polyacrylamide based stationary phase was used in hydrophilic-interaction-chromatography (HILIC) mode to purify APTS labeled glycans. The whole sample preparation procedure can be performed at the 96-well-plate level with a hands-on time of less than 2.5 h. Purified APTS-labeled N-glycans were analyzed using a standard capillary DNA sequencer. The optimized sample preparation, combined with robust, HR, high-sensitive multiplexed CGE-LIF based measurement and fully automated data evaluation, enables highly reproducible “real” HTP analysis of protein N-glycosylation. 1. Schwarzer J, Rapp E, Reichl U, Electrophoresis, 2008, 29, 4203-4214. 2. Ruhaak LR, Hennig R, Huhn C, Borowiak M, Dolhain RJEM, Deelder AM, Rapp E, Wuhrer M, Journal of Proteome Research, 2010, 9,6655-6664.
