Protein modification, glycosylation

Protein modification 36 Variable Acetylation, fatty acylation, glycosylation, methylation, phosphorylation, ADP-ribosylation, ubiquitination, proteolytic digestion... 

Most frequently, extracts of either prokaryotic or eukaryotic origin as such from Escherichia coli, wheat germ or rabbit reticulocytes are employed for cost reasons and availability. While those based on E. coli are unable of post-translational protein modification, eukaryotic extracts do allow synthesis of glycosylated or phosphorylated proteins to some extent when additional components, such as microsomes for glycosylation are added. Care needs to be taken with cell-free systems recombinated from the individual components when a native protein is to be produced that does not fold spontaneously... 

S. P. Wolff and R. T. Dean, Glucose autooxidation and protein modification. The potential role of autooxidative glycosylation in diabetes, Biochem. J., 245 (1987) 243-250. 

While proteins are synthesized on ribosomes the translation product is often subject to considerable post-translational modification that can involve proteolytic processing of the initial proprotein and covalent modification of the processed protein by glycosylation (addition of sugar residues), acylation (e.g. with fatty acids), methylation and phosphorylation. Unconjugated proteins are those in which there is no non-amino acid substituent and conjugated proteins are those that have been modified with non-amino acid entities. 

Shakoori AR. Phosphorylation and glycosylation interplay Protein modifications at hydroxy amino acids and prediction of signaling functions of the human beta(3) integrin family. J. Cell Biochem. 2006 99 706-718. 41. 

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. 

---