Isolation of Glycoproteins and Identification of Their N-Linked Glycosylation Sites

Protein glycosylation has long been recognized as a very common posttranslational modifi- cation. Protein glycosylation is prevalent in proteins destined for extracellular environments. These include proteins localized on the extracellular surface and those secreted to body fluids. In search of a method that has the potential to identify and quantify most proteins found in body fluids or the cell surface, we have recently developed a novel method for solid-phase extraction of formerly
N
-linked glycosylated peptides from glycoproteins. It has been shown that proteins secreted to body fluids or localized on the cell surface can be specifically enriched by this method. The technique is based on the conjugation of glycoproteins to a solid support using hydrazide chemistry, removal of nonglycosylated peptides by trypsin digestion, stable isotope labeling of glycopeptides, and the specific release of formerly
N
-linked glycosylated peptides via peptide-
N
-glycosidase. The recovered formerly
N
-linked glycopeptides are then identified and quantified by tandem mass spectrometry.