title: Determination of glycosylation sites and disulfide bond structures using LC/ESI-MS/MS analysis.
abstract: Significant progress has been made in discovering and cloning a host of proteins, including a range of glycoproteins. The availability of their predicted amino acid sequences provides useful information, including potential N-linked glycosylation sites. However, only a limited number of protein structures have been solved, and very little is known about the structures of membrane proteins. One of the important structural elements of a protein is its disulfide bonds. These covalent bonds place conformational constraints on the overall protein structure, and thus, their identification provides important structural information. A second important posttranslational modification found in proteins is N-linked glycosylation. Although potential sites of N-linked glycosylation can be predicted from a protein's primary sequence based on the presence of N-X-S/T sequences, not all of the predicted sites will be glycosylated. Therefore, N-linked glycosylation sites must be located by structural analysis. We have developed a simple and sensitive method for determining the presence of free cysteine (Cys) residues and disulfide-bonded Cys residues, as well as the N-linked glycosylation sites in glycoproteins by liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) in combination with protein database searching using the programs Sequest and Mascot. The details of our method are described in this chapter.
authors.affiliation: Department of Chemistry and Biochemistry, San Francisco State University, CA, USA.
authors.forename: Ten-Yang
authors.initials: TY
authors.lastname: Yen
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Macher
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides; D006031:Glycosylation; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D004220:Disulfides; D010455:Peptides; D003545:Cysteine