Glycoprotein characterization combining intact protein and glycan analysis by capillary electrophoresis-electrospray ionization-mass spectrometry.

Full 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 alpha1-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.

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Author information

Author/s: Balaguer, Elvira (E); Neusüss, Christian (C);

Affiliation: University of Barcelona, Barcelona, Spain.

Journal and publication information

Publication Type: Journal Article

Journal: Analytical chemistry (Anal Chem), published in United States. (Language: eng)

Reference: 2006-Aug; vol 78 (issue 15) : pp 5384-93

Dates: Created 2006/08/01; Completed 2007/06/04;

PMID: 16878873, status: MEDLINE (last retrieval date: 12/26/2008)

Sourced from the National Library of Medicine. Abstract text and other information may be subject to copyright.

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MeSH headings (categories)

This article was linked to the MESH Headings shown below.

Electrophoresis, Capillary - methods
Glycoproteins - analysis
Polysaccharides - analysis

Proteins - analysis
Sensitivity and Specificity
Spectrometry, Mass, Electrospray Ionization - methods

Associated Chemicals: Glycoproteins (0) ; Polysaccharides (0) ; Proteins (0)

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