A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation

doi:10.1073/pnas.2335201100

A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation

^*Center for New Directions in Organic Synthesis, Departments of ^†Chemistry and ^‡Molecular and Cell Biology, and ^§Howard Hughes Medical Institute, University of California, Berkeley, CA 94720-1460

Edited by Kai Simons, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany, and approved September 11, 2003 (received for review August 13, 2003)

Abstract

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-α-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.

Footnotes

↵ ¶ To whom correspondence should addressed. E-mail: bertozzi{at}cchem.berkeley.edu.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: GalNAc, N-acetylgalactosamine; ppGalNacTs, polypeptide N-acetyl-α-galactosaminyltransferases; pNGase-F, peptide N-glycosidase F; GalNAz, N-azidoacetylgalactosamine; 2AzGal, 2-azido-2-deoxygalactose; CHO, Chinese hamster ovary; Gal, galactose.