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

TLDR

Mucin‑type O‑linked glycoproteins mediate diverse biological interactions, are synthesized by ppGalNAcTs, and lack a consensus sequence, complicating prediction from primary sequence. The authors developed a chemical‑tagging method that incorporates an azide into mucin‑type O‑linked glycoproteins, enabling selective covalent modification from complex cell lysates. The azide functions as a bioorthogonal handle that can be coupled to probes via Staudinger ligation for selective labeling. Using the azido‑GalNAc analog GalNAz, the authors demonstrated efficient metabolic labeling of mucin‑type O‑linked glycoproteins in multiple cell types, validated on a recombinant glycoprotein, and showed that this approach facilitates proteomic identification of endogenous O‑linked glycoproteins.

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.