2004/01/29 by Jakob Bunkenborg, Bartosz Pilch, Alexandre V. Podtelejnikov +1 · 2 citations
Biochemistry, Genetics and Molecular Biology · Chemistry · #Glycosylation and Glycoproteins Research #RNA and protein synthesis mechanisms #Advanced Proteomics Techniques and Applications
paper · doi:10.1002/pmic.200300556
openalex publication_date 2004/01/29 · openalex created_date 2016/06/24 · openalex updated_date 2026/08/01
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.