文章基本信息

标题：A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation
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作者：Howard C. Hang ; Chong Yu ; Darryl L. Kato 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2003
卷号：100
期号：25
页码：14846-14851
DOI：10.1073/pnas.2335201100
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Mucin-type O-linked glycoproteins are involved in a variety of biological interactions in higher eukaryotes. The biosynthesis of these glycoproteins is initiated by a family of polypeptide N-acetyl--galactosaminyltransferases (ppGalNAcTs) that modify proteins in the secretory pathway. The lack of a defined consensus sequence for the ppGalNAcTs makes the prediction of mucin-type O-linked glycosylation difficult based on primary sequence alone. Herein we present a method for labeling mucin-type O-linked glycoproteins with a unique chemical tag, the azide, which permits their selective covalent modification from complex cell lysates. From a panel of synthetic derivatives, we identified an azido GalNAc analog (N-azidoacetylgalactosamine, GalNAz) that is metabolized by numerous cell types and installed on mucin-type O-linked glycoproteins by the ppGalNAcTs. The azide serves as a bioorthogonal chemical handle for selective modification with biochemical or biophysical probes using the Staudinger ligation. The approach was validated by labeling a recombinant glycoprotein that is known to possess O-linked glycans with GalNAz. In addition, GalNAz efficiently labeled mucin-type O-linked glycoproteins expressed at endogenous levels. The ability to label mucin-type O-linked glycoproteins with chemical tags should facilitate their identification by proteomic strategies.