Title : Screening for
N-glycosylated proteins by liquid chromatography mass spectrometry
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