Introductory Glycosylation Analysis Using SDS-PAGE and Peptide Mass Fingerprinting

Abstract

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.

Characterization of two new monoclonal antibodies against human papillomavirus type 16 L1 protein

“…CDR3 encoded by the VH gene of two mAb had 9 amino acids, which suggested that the corresponding antigens were relatively complex [12]. The VH of AE3 mAb was rich in serine, threonine, and asparagine residues, which are potential glycosylation sites [13]. Although the VH of AG7 mAb had a greater number of variant amino acids compared to the VH of AE3 mAb, there were still some serine, threonine, and asparagine residues in the VH of AG7 mAb, which may enhance the hydrophilicity and the affinity of the antibody to corresponding antigen [14].…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2009–2010

“…Experimental details for peptide mass fingerprinting and identification of glycosylation sites have been published by Wilson, Simpson, and Cooper-Liddell (2009).…”

Review Soybeans as bioreactors for biopharmaceuticals and industrial proteins

“…For these purposes, MS is the most versatile and broad analytical tool available in biochemistry for determining the mass in Daltons of recombinant proteins. It can be also used for sequence determination by de novo sequencing (Shevchenko et al, 2006), and for determination of post-translational modifications, such as phosphorylation (Hunter and Games, 1994), glycosylation (Wilson et al, 2009) and protein oxidation (Barnes et al, 2008). In addition to MS methods, N-terminal sequencing can be verified by the EDMAN degradation technique when it cannot be sequenced by MS due to ionization problems or a long N-terminal fragment (Edman, 1949).…”