Screening for N-glycosylated proteins by liquid chromatography mass spectrometry.
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.
Bunkenborg
Jakob
J
MDS Proteomics Odense, Denmark.
Pilch
Bartosz J
BJ
Podtelejnikov
Alexandre V
AV
Wiśniewski
Jacek R
JR
D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000032:analysis* / Q000737:chemistry; D002853:Chromatography, Liquid; D003208:Concanavalin A / Q000737:chemistry*; D006023:Glycoproteins / Q000032:analysis* / Q000097:blood / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis* / Q000097:blood / Q000737:chemistry; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D014909:Wheat Germ Agglutinins / Q000737:chemistry*
D001798:Blood Proteins; D006023:Glycoproteins; D009844:Oligosaccharides; D014909:Wheat Germ Agglutinins; D003208:Concanavalin A; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase