| Online nanoliquid chromatography-mass spectrometry and nanofluorescence detection for high-resolution quantitative N-glycan analysis. | |
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MedLine Citation:
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PMID: 22330744 Owner: NLM Status: Publisher |
Abstract/OtherAbstract:
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The characterization of the repertoire of glycans at the quantitative and qualitative levels on cells and glycoproteins is a necessary step to the understanding of glycan functions in biology. In addition, there is an increasing demand in the field of biotechnology for the monitoring of glycosylation of recombinant glycoproteins, an important issue with regard to their safety and biological activity. The enzymatic release followed by fluorescent derivatization of glycans and separation by normal phase high-performance liquid chromatography (HPLC) has proven for many years to be a powerful approach to the quantification of glycans. Characterization of glycans has classically been performed by mass spectrometry (MS) with external standardization. Here, we report a new method for the simultaneous quantification and characterization of the N-glycans on glycoproteins without the need for external standardization. This method, which we call glycan nanoprofiling, uses nanoLC-coupled electrospray ionization (ESI)-MS with an intercalated nanofluorescence reader and provides effective single glycan separation with subpicomolar sensitivity. The method relies on the isolation and coumaric derivatization of enzymatically released glycans collected by solid phase extraction with porous graphitized carbon and their separation over polyamide-based nanoHPLC prior to serial nanofluorescence and nanoelectrospray mass spectrometric analysis. Glycan nanoprofiling is a broadly applicable and powerful approach that is sufficient to identify and quantify many glycan oligomers in a single run. Glycan nanoprofiling was successfully applied to resolve the glycans of monoclonal antibodies, showing that this method is a fast and sensitive alternative to available methods. |
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Authors:
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Hakan Kalay; Martino Ambrosini; Patrick H C van Berkel; Paul W H I Parren; Yvette van Kooyk; Juan J García Vallejo |
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Publication Detail:
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Type: JOURNAL ARTICLE Date: 2012-1-28 |
Journal Detail:
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Title: Analytical biochemistry Volume: 423 ISSN: 1096-0309 ISO Abbreviation: - Publication Date: 2012 Jan |
Date Detail:
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Created Date: 2012-3-2 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 0370535 Medline TA: Anal Biochem Country: - |
Other Details:
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Languages: ENG Pagination: 153-162 Citation Subset: - |
Copyright Information:
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Copyright © 2012 Elsevier Inc. All rights reserved. |
Affiliation:
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Department of Molecular Cell Biology and Immunology, Faculty of Medicine, VU University Medical Center, 1007 MB Amsterdam, The Netherlands. |
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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