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J Biomol Tech. 2010 September; 21(3 Suppl): S24.
PMCID: PMC2918142

Characterization of Neutral and Acidic Glycopeptides by ZIC-HILIC Enrichment and Mass Spectrometry

J. Wohlgemuth,1 S. Andrecht,1 A. Schneider,2 and A. Resemann2
1Merck KGaA, Darmstadt, Germany;
2Bruker Daltonik GmbH, Bremen, Germany

Abstract

RP-14

Glycosylation is the most abundant protein posttranslational modification and is involved in many relevant biological processes and crucial to the understanding of many diseases. In depth analysis of glycosylation sites is difficult, however, as glycopeptides exhibit a significant micro heterogeneity at glycosylation sites. In addition, ion suppression effects require selective methods for glycopeptide enrichment. Mass spectrometric analysis of the two distinct glycopeptide moieties is challenging because both the peptide as well as the glycan moiety have to be elucidated for a full structural assignment. We used Fetuin, Alpha-1-Acidglycoprotein and Asialo-Fetuin to equally representing sialylated and non-sialylated glycosylic structures. In addition, monoclonal antibodies were analyzed as a dedicated example for pharmaceutical QC. Samples were digested with trypsin and glycopeptides were enriched using a dedicated ZIC glycocapture resin in combination with an optimized buffer system (EMD Chemicals Inc.). Subsequently, the glycopeptides were analyzed using ESI ion trap MS for glycoprofiling and MALDI-TOF/TOF-MS for in depth characterization of the glycopeptides. In MALDI-TOF/TOF instruments, N-linked glycopeptides undergo an indicative cross-ring fragmentation of the GlcNAc that links to the Asn. Indicative fragments allow obtaining the peptide mass safely as well as the glycan composition. Subsequent Mascot searches of the glycopeptide MS/MS spectra provided for the sequence of the glycopeptide and the localization of the glycosylation site. Searches in glycan databases based on the same glycopeptide MS/MS spectra allowed to complete the characterization of N-linked glycopeptides. For the first time, MALDI-TOF/TOF-MS analysis of multi-sialylated glycopeptides was shown after linear positive ion mode detection of the respective precursor ions. In comparison to direct MS analysis of glycoprotein digests, the enriched samples allowed to detect more glycopeptides and permitted the acquisition of MS/MS spectra of higher quality. Additional separation techniques prior to MS analysis such as RP-HPLC may allow even greater insights into the complexity of protein glycosylation.


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