Protein Glycosylation and Phosphorylation

1 Glycosylation. Carbohydrate modifications of proteins (glycosylation) are key factors in modulating protein structures and functions within cells. Glycosylation affects probably more than half of all proteins in a eukaryotic cell [40]. In the extracellular environment, the oligosaccharide moieties of 

Removal of glycans through P-elimination for O-linked glycans or enzy- 

Top-down sequence analysis of whole glycoprotein ions using CID and ion/ion proton transfer in a quadrupole ion trap MS [66], This approach eliminated gas-phase deglycosylation of N-linked oligosaccharide in ribonuclease B, and the glycosylation site was identified to be Asn-Leu-Thr at residues 34-36 [66], 

In order to assess the size of the carbohydrate component, deglycosylated CHO IL-4 by A-glycanase was analyzed by ESI-MS. The deconvoluted spectrum displayed an MW of 14,955 Da, which conformed well to the theoretical MW of the protein (MW 14,963 Da) when considering the presence of three disulfide bonds in the protein. 

Mapping of the primary structure of CHO IL-4 was carried out by tryptic hydrolysis followed by measurement of the resulting peptide fragments by on-line HPLC/ESI-MS. Since the primary sequence of IL-4 was known, this MS mapping approach could confirm the cDNA-derived protein sequence, 

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Whelan SA, Hart GW. Proteomic approaches to analyze the dynamic relationships between nucleocytoplasmic protein glycosylation and phosphorylation. Circ. Res. 2003 93 1047-1058. Konrad RJ, Zhang F, Hale JE, Knierman MD, Becker GW, Kudlow JE. Alloxan is an inhibitor of the enzyme O-linked N-acetylglucosamine transferase. Biochem. Biophys. Res. Commun. 2002 293 207-212. 

Our initial applications of the stepped collision energy LC-ESMS approach involved selective detection (and differentiation) of N- or O-glycosylated peptides (5,6), and detection of phosphorylated peptides (7,8,11). The method has been in routine use in our laboratory for over two years and during this time has become one of the mainstays of our work in characterizing protein modifications. Here we present some of our more recent studies on protein glycosylation and phosphorylation, and illustrate a preliminary evaluation of the stepped collision energy LC-ESMS method for selective detection of sulfated and acylated peptides in protein digests. 

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