Quantitative site-specific analysis of protein glycosylation by LC-MS using different glycopeptide-enrichment strategies
| Autor: | Michael Karas, Jessica Wohlgemuth, Thomas Eichhorn, Sven Andrecht, Robertus Hendriks |
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| Rok vydání: | 2009 |
| Předmět: |
Spectrometry
Mass Electrospray Ionization Glycan Glycosylation Biophysics Neuraminidase Ion suppression in liquid chromatography–mass spectrometry Peptide Biochemistry chemistry.chemical_compound Liquid chromatography–mass spectrometry Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase ortho-Aminobenzoates Molecular Biology Chromatography High Pressure Liquid Titanium chemistry.chemical_classification Chromatography biology Hydrophilic interaction chromatography Glycopeptides Cell Biology Glycopeptide Hydrazines chemistry biology.protein Quantitative analysis (chemistry) |
| Zdroj: | Analytical Biochemistry. 395:178-188 |
| ISSN: | 0003-2697 |
| DOI: | 10.1016/j.ab.2009.08.023 |
| Popis: | A common technique for analysis of protein glycosylation is HPLC coupled to mass spectrometry (LC-MS). However, analysis is challenging due to a low abundance of glycopeptides in complex protein digests, microheterogeneity at the glycosylation site, ion suppression effects, and competition for ionization by coeluting peptides. Specific sample preparation is necessary for a comprehensive and site-specific glycosylation analysis by MS. In this study we qualitatively compared hydrophilic interaction chromatography (HILIC) and hydrazine chemistry for the enrichment of all N-linked glycopeptides and titanium dioxide for capturing sialylated glycopeptides from a complex peptide mixture. Bare silica, microcrystalline cellulose, amino-, amide- (TSKgel Amide-80), and sulfobetaine-(ZIC-HILIC) bonded phases were evaluated for HILIC enrichment. The experiments revealed that ZIC-HILIC and TSKgel Amide-80 are very specific for capturing glycopeptides under optimized conditions. Quantitative analysis of N-glycosidase F-released and 2-aminobenzamide-labeled glycans of a ZIC-HILIC-enriched monoclonal antibody demonstrated that glycopeptides could be enriched without bias for particular glycan structures and without significant losses. Sialylated glycopeptides could be efficiently enriched by titanium dioxide and in addition to HILIC both methods enable a comprehensive analysis of protein glycosylation by MS. Enrichment of N-linked glycopeptides by hydrazine chemistry resulted in lower peptide recovery using a more complex enrichment scheme. |
| Databáze: | OpenAIRE |
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