Screening for N-glycosylated proteins by liquid chromatography mass spectrometry.

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Bunkenborg J, Pilch BJ, Podtelejnikov AV, Wisniewski JR

Screening for N-glycosylated proteins by liquid chromatography mass spectrometry.

Proteomics. 2004 Feb;4(2):454-65.

PubMed ID
14760718 [ View in PubMed
]
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.

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Polypeptides
NameUniProt ID
ProthrombinP00734Details
Corticosteroid-binding globulinP08185Details
Antithrombin-IIIP01008Details
TransthyretinP02766Details
SerotransferrinP02787Details
Alpha-2-macroglobulinP01023Details
Alpha-1-acid glycoprotein 1P02763Details
Serum paraoxonase/arylesterase 1P27169Details
Alpha-1-antitrypsinP01009Details
Insulin-like growth factor-binding protein 3P17936Details
Kininogen-1P01042Details
von Willebrand factorP04275Details
VitronectinP04004Details
CeruloplasminP00450Details
FibronectinP02751Details
Fibrinogen gamma chainP02679Details
Plasma serine protease inhibitorP05154Details
CholinesteraseP06276Details
Plasma protease C1 inhibitorP05155Details
Beta-2-glycoprotein 1P02749Details
Zinc-alpha-2-glycoproteinP25311Details
Complement C3P01024Details
Carboxypeptidase N subunit 2P22792Details
Alpha-1-acid glycoprotein 2P19652Details
Ig alpha-2 chain C regionP01877Details
Coagulation factor XIIP00748Details
Complement C4-AP0C0L4Details
Complement C4-BP0C0L5Details
Apolipoprotein DP05090Details
Ig mu chain C regionP01871Details
AfaminP43652Details
Alpha-1B-glycoproteinP04217Details
Alpha-2-HS-glycoproteinP02765Details
C4b-binding protein alpha chainP04003Details
Complement component C9P02748Details
Complement factor HP08603Details
Complement factor IP05156Details
ClusterinP10909Details
Leucine-rich alpha-2-glycoproteinP02750Details
LumicanP51884Details
C4b-binding protein beta chainP20851Details
Inter-alpha-trypsin inhibitor heavy chain H1P19827Details
Inter-alpha-trypsin inhibitor heavy chain H4Q14624Details
Alpha-1-antichymotrypsinP01011Details
Apolipoprotein B-100P04114Details
HemopexinP02790Details
HaptoglobinP00738Details