Introductory glycosylation analysis using SDS-PAGE and peptide mass fingerprinting.
Glycosylation is extremely complex, with the potential for a protein to have oligosaccharides attached at multiple sites, and for each site of glycosylation to have multiple structures attached to it. Structural information on the oligosaccharides bound to either asparagine residues (N-linked) or serine and threonine residues (O-linked) requires sensitive, specialised, and complex techniques and equipment. We show here, however, that a large amount of information regarding the glycosylation of glycoproteins can be obtained with common protein techniques such as 1D SDS-PAGE and peptide mass fingerprinting (PMF). Enzymatic deglycosylation in combination with SDS-PAGE and PMF analysis can determine the relative percentage of N-linked carbohydrate on the glycosylated protein, as well as attachment sites of the oligosaccharides.
Wilson
Nicole
N
Apollo Cytokine Research, Beaconsfield, Sydney, NSW, Australia.
Simpson
Raina
R
Cooper-Liddell
Catherine
C
D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004798:Enzymes / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D010449:Peptide Mapping / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D004798:Enzymes; D006023:Glycoproteins