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Abstract No.: TuP-186
Session: Posttranslational Modifications
Presentation date: Tue, Aug 29, 2006
Presentation time: 14:30 – 16:00

Capillary Zone Electrophoresis Coupled to Electrospray and Matrix-Assisted Laser Desorption/Ionisation Mass Spectrometry for the Analysis of Glycopeptides

Sabine Amon1, Alexander Plematl1, Andreas Rizzi1

1 Institute of Analytical Chemistry, University of Vienna, Vienna, Austria

Correspondence address: Sabine Amon, Institute of Analytical Chemistry, University of Vienna, Waehringerstrasse 38, Vienna, 1090 Austria.

Keywords: Electrophoresis Capillary Zone; Glycoproteins; Mass Spectrometry, Capillary Electrophoresis; MS/MS, Capillary Electrophoresis.

Novel aspect: Analysis of glycopeptides by on-line capillary zone electrophoresis electrospray mass spectrometry and automated off-line capillary zone electrophoresis matrix-assisted laser desorption/ionisation mass spectrometry.

 

Objectives
The analysis and characterisation of glycosylation in proteins and peptides is of great interest due to the significant impact of glycosylation on function, half-life and immunogenicity. This is mostly accomplished by analysing glycopetides (obtained by digestion of glycoproteins) by mass spectrometry. Since desorption/ionisation of glycosylated peptides is suppressed in the presence of non-glycosylated ones, a pre-separation step is recommended. Most commonly this is done by RP-HPLC where, however, the analysis of glycopeptides with small peptide backbones and large glycans eluted near the void volume is problematic. A promising, complementary method is capillary zone electrophoresis (CZE) coupled to ESI- or MALDI-MS. In this approach, a high and stable EOF is required for the spray and spotting process, respectively, which is not provided by conventional fused silica capillaries. In this work different capillary coatings along with background electrolytes of different pH values were evaluated for the purpose of a complete and comprehensive analysis of glycopeptides in a mixture of tryptic peptides.

Methods
The reduced, alkylated and tryptically digested glycoproteins were separated by CZE using fused silica capillaries coated by hexadimethrine bromide or additionally dextran sulfate. Along with background electrolyte solutions of various pH values different selectivities regarding the glycopeptides could be achieved. CZE was on-line coupled to an ESI-QIT-MS using a triple tube sprayer supplying sheath-flow. Automated off-line coupling to MALDI-MS was performed by a roboting device for sheath-flow-assisted spotting onto a MALDI-target. Analysis of the deposited spots was carried out on a linear MALDI-TOF-MS as well as on a multistage MALDI-QIT-rTOF-MS.

Results
The selection of the capillary coating and the background electrolyte solution allows one (i) to separate glycopeptides from the other peptides present in the tryptic digest a step necessary to avoid suppression of glycopeptide ionisation in the presence of non-glycosylated peptides and (ii) to separate the glycopeptides from each other being advantageous for on-line multistage MS. On-line CZE-ESI-MS and robot-assisted off-line CZE-MALDI-MS were both demonstrated to give reliable results in terms of peptide coverage (singlestage MS) and structure information (multistage MS). The different benefits of the applied approaches were examined.

Conclusions
A sufficient ionisation yield for glycopeptides in ESI- as well as MALDI-MS without suffering from suppression effects requires pre-separation of the glycopeptides from most of the other sample components. For this purpose CZE under controlled electroosmotic flow conditions was successfully applied and proved to be beneficial for analysis time, robustness of the CE-MS system, stability of transfer of the analytes to the MS and completeness of glycopeptides detected and characterised.