17th International Mass Spectrometry Conference :: Prague, 2006
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|Session:||Glycomics and Oligosaccharides|
|Presentation date:||Mon, Aug 28, 2006|
|Presentation time:||14:30 – 16:00|
Yann Hebert1, Kathrin Sparbier2, Markus Kostrzewa2, Thomas Wenzel2, Irina Kessler2, Arndt Asperger21 Bruker Daltonique, Wissembourg, France
Correspondence address: Yann Hebert, Bruker Daltonique, 34, rue de l' industrie, Wissembourg, 67160 France.
Web site: http://www.bdal.de
Keywords: Chromatography, Liquid -, High Pressure (HPLC); Glycoproteins; MALDI; Mass Spectrometry, Time of Flight.
Novel aspect: Combination of magnetic beads for fractionation and offline LC-maldi for the more detailed analyses of glycosylation in complex biological samples.
In the course of comprehensive proteomic analyses, high sample complexity and the enormous dimensions of the concentration range to be covered are the main difficulties. Thus, direct analysis of post-translationally modified peptides and proteins is often impossible. Selective enrichment and purification of peptides and proteins can be attained by chromatographic methods as supported by specifically functionalized magnetic particles. Glycoproteins and-peptides can be isolated by lectin affinity chromatography or covalent binding to boronic acids. According to the type of glycosylation and the presented carbohydrate motif, binding to different functionalities is favored. Human serum contains a high number of glycoprotein species comprising several orders of magnitude in concentration. Thereby, isolation and subsequent identification of low abundant glycoproteins from complex serum samples is a challenging task.
Human serum (20 µl) was incubated with different magnetic microparticles functionalized with Concanavalin A (ConA), wheat germ agglutinin (WGA) and boronic acids, respectively. Isolated proteins were released from the beads under acidic conditions, dried and subsequently re-dissolved and digested with trypsin over night at 37°C. The resulting complex mixture of peptides was separated by LC on a PepMap C18 column applying a gradient from zero to 45 % acetonitrile within 110 min. 384 fractions were directly spotted on a disposable AnchorChip target pre-spotted with HCCA matrix and analyzed by an Ultraflex II TOF/TOF.
Applying this fast and simple approach, 101 proteins were identified from human serum comprising 84 known glycosylated proteins. According to the specific binding preferences of the different types of beads, complementary results were obtained from experiments using magnetic ConA-, WGA- and boronic acids beads, respectively. An overlap of 14 glycoproteins was detected by all three bead functionalities together. The two lectins in use, ConA and WGA, as expected, showed a certain similarity in their binding behavior, revealing 10 shared glycoproteins. In contrast, the overlap of the identified glycoproteins from WGA and ConA with boronic acids was only 3 and 5 proteins, respectively. The fraction of glycoproteins uniquely captured by boronic acid beads comprised four proteins with C-linked glycosylation.
The approach described here could be extended to the more detailed analyses of glycosylation in complex biological samples in the sense of mapping N-glycosylation sites or full characterisation of glycane structures.