Glycoprotein characterization combining intact protein and glycan analysis by capillary electrophoresis-electrospray ionization-mass spectrometry
Glycoprotein characterization combining intact protein and glycan analysis by capillary electrophoresis-electrospray ionization-mass spectrometry
Engineering Village 2
2006 Elsevier Inc.
Accession number: 063210059487
Title: Glycoprotein characterization combining intact protein and glycan analysis by capillary electrophoresis-electrospray ionization-mass spectrometry
Authors: Balaguer, Elvira; Neususs, Christian
Author affiliation: Bruker Daltonik GmbH, 28359 Bremen, Germany
Serial title: Analytical Chemistry
Abbreviated serial title: Anal. Chem.
Volume: v 78
Issue: n 15
Issue date: Aug 1 2006
Publication year: 2006
Pages: p 5384-5393
Language: English
ISSN: 0003-2700
CODEN: ANCHAM
Document type: Journal article (JA)
Publisher: American Chemical Society, Columbus, OH 43210-3337, United States
Abstract: Glycosylated proteins play important roles in a large number of biological processes. Therefore, a complete characterization in terms of glycan structures and glycoform heterogeneity is needed. In this paper, a combined approach based on glycan and intact glycoprotein analysis by capillary zone electrophoresis-electrospray-mass spectrometry (CZE-ESI-MS) is presented. Based on a new capillary coating, a CZE-ESI-MS method for the separation and characterization of intact glycoproteins has been developed and compared to a method recently introduced for the characterization of erythropoietin. The excellent glycoform separation results in high-quality mass spectra, high dynamic range, and good sensitivity, allowing the correct characterization of minor glycan modifications. Additionally, a CZE-ESI-MS separation method for underivatized N-glycans has been developed. The separation of glycans differing in the degree of sialic acids and repeats of noncharged carbohydrates is achieved. The separation power of the method is demonstrated by obtaining mobility differences in glycans differing only by 16 Da. A time-of-flight mass spectrometer allowed the correct identification of the glycan composition based on high mass accuracy and resolution, identifying even minor modifications such as the exchange of "O" by "NH". An ion trap mass spectrometer provided structural information of the underivatized glycans from fragmentation spectra. The general applicability of both methods to glycoprotein analysis is illustrated for erythropoietin, fetuin, and a1-acid glycoprotein. The results obtained by the glycan analysis allowed an unequivocal glyco-assignment to the masses obtained for the intact proteins as long as the protein backbone is well characterized. Furthermore, modifications found for intact proteins can be attributed to differences in the glycostructure. ? 2006 American Chemical Society.
Number of references: 32
Ei main heading: Proteins
Ei controlled terms: Electrophoresis - Ionization - Mass spectrometry - Separation - Carbohydrates
Uncontrolled terms: Glycan structures - Glycoform heterogeneity - Glycoproteins - Capillary coatings - Mass accuracy
Ei classification codes: 804.1 Organic Compounds - 701.1 Electricity: Basic Concepts & Phenomena - 801.3 Colloid Chemistry - 801.4.1 Electrochemistry - 802.2 Chemical Reactions - 802.3 Chemical Operations
Treatment: Experimental (EXP)
DOI: 10.1021/ac060376g
Database: Compendex
Compilation and indexing terms, ? 2006 Elsevier Inc. All rights reserved
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