Screening for N-glycosylated proteins by liquid chromatography mass spectrometry

Jakob Bunkenborg, Bartosz J Pilch, Alexandre V Podtelejnikov, Jacek R Wiśniewski

171 Citationer (Scopus)

Abstract

In the last few years mass spectrometry has become the method of choice for characterization of post-translationally modified proteins. Whereas most protein chemical modifications are binary in the sense that only one change can be associated with a given residue, many different oligosaccharides can be attached to a glycosylation site residue. The detailed characterization of glycoproteins in complex biological samples is extremely challenging. However, information on N-glycosylation can be gained at an intermediary level. Here we demonstrate a procedure for mapping N-glycosylation sites in complex mixtures by reducing sample complexity and enriching glycoprotein content. Glycosylated proteins are selected by an initial lectin chromatography step and digested with endoproteinase Lys-C. Glycosylated peptides are then selected from the digest mixture by a second lectin chromatography step. The glycan components are removed with N-glycosidase F and the peptides digested with trypsin before analysis by on-line reversed-phase liquid chromatography mass spectrometry. Using two different lectins, concanavalin A and wheat germ agglutinin, this procedure was applied to human serum and a total of 86 N-glycosylation sites in 77 proteins were identified.
OriginalsprogEngelsk
TidsskriftProteomics
Vol/bind4
Udgave nummer2
Sider (fra-til)454-65
Antal sider12
ISSN1615-9853
DOI
StatusUdgivet - 2004
Udgivet eksterntJa

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