Rapid release of n-linked glycans from glycoproteins by pressure-cycling technology

Zoltan Szabo, A. Guttman, Barry L. Karger

Research output: Contribution to journalArticle

49 Citations (Scopus)

Abstract

The standard, well-established sample preparation protocol to release N-linked glycans from glycoproteins for downstream analysis requires relatively long deglycosylation times (from several hours to overnight) and relatively high endoglycosidase concentration (from 1:250 to 1:500 enzyme:substrate molar ratio). In this paper, we significantly improve this standard protocol by the use of pressure-cycling technology (PCT) to increase the speed and decrease the relative amount of PNGase F during the release of N-linked glycans from denatured glycoproteins. With the application of pressure cycling from atmospheric to as high as 30 kpsi, >95% release of the asparaginelinked glycans from bovine ribonuclease B, human transferrin, and polyclonal human immunoglobulin was rapidly achieved in a few minutes using as low as 1:2500 enzyme: substrate molar ratio. The deglycosylation rate was first examined by SDS-PAGE at the protein level. The released glycans were then quantitated by capillary electrophoresis with laser induced fluorescence detection (CE-LIF). This new sample preparation protocol readily supports largescale glycan analysis of biopharmaceuticals with rapid deglycosylation times.

Original languageEnglish
Pages (from-to)2588-2593
Number of pages6
JournalAnalytical Chemistry
Volume82
Issue number6
DOIs
Publication statusPublished - Mar 15 2010

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Polysaccharides
Glycoproteins
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Capillary electrophoresis
Glycoside Hydrolases
Substrates
Enzymes
Transferrin
Immunoglobulins
Fluorescence
Lasers
Proteins

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Rapid release of n-linked glycans from glycoproteins by pressure-cycling technology. / Szabo, Zoltan; Guttman, A.; Karger, Barry L.

In: Analytical Chemistry, Vol. 82, No. 6, 15.03.2010, p. 2588-2593.

Research output: Contribution to journalArticle

Szabo, Zoltan ; Guttman, A. ; Karger, Barry L. / Rapid release of n-linked glycans from glycoproteins by pressure-cycling technology. In: Analytical Chemistry. 2010 ; Vol. 82, No. 6. pp. 2588-2593.
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