Large-scale site-specific mapping of the O-GalNAc glycoproteome

Abstract

Protein glycosylation is one of the most common protein modifications. A major type of protein glycosylation is O-GalNAcylation, in which GalNAc-type glycans are attached to protein Ser or Thr residues via an O-linked glycosidic bond. O-GalNAcylation is thought to play roles in protein folding, stability, trafficking and protein interactions, and identification of the site-specific O-GalNAc glycoproteome is a crucial step toward understanding the biological significance of the modification. However, lack of suitable methodology, absence of consensus sequon of O-GalNAcylation sites and complex O-GalNAc glycan structures pose analytical challenges. We recently developed a mass spectrometry-based method called extraction of O-linked glycopeptides (EXoO) that enables large-scale mapping of site-specific mucin-type O-GalNAcylation sites. Here we provide a detailed protocol for EXoO, which includes seven stages of: (1) extraction and proteolytic digestion of proteins to peptides, (2) sequential guanidination and de-salting of peptides, (3) enrichment of glycopeptides, (4) solid-phase peptide conjugation and release of O-GalNAc glycopeptides using the OpeRATOR protease, (5) liquid chromatography with tandem mass spectrometry analysis of O-GalNAc glycopeptides, (6) identification of O-GalNAc glycopeptides by database search and (7) quantification of O-GalNAc glycopeptides. Using this protocol, thousands of O-GalNAcylation sites from hundreds of glycoproteins with information regarding site-specific O-GalNAc glycan can be identified and quantified from complex samples. The protocol can be performed by a researcher with basic proteomics skills and takes about 4 d to complete.

Figure 1 |

Workflow diagram of EXoO protocol and O-GalNAcylation pathway for Gal(1)GalNAc(1)Sia(2). (a) Schematic representation of EXoO method. Image is adapted and from ref.16. (b) Steps of EXoO protocol. (c) O-GalNacylation pathway for Gal(1)GalNAc(1)Sia(2).

Figure 2 |

MS/MS spectra of site-specific O-GalNAc glycopeptides. The glycopeptides are generated using the protocol. O-GalNAc glycosylation sites are at the N-termini of peptide sequences. Different glycan compositions are showed to illustrate the approach for defining site-specific O-GalNAc glycans. (a) MS/MS spectra of a glycopeptide with Hex(1)HexNAc(1). (b) MS/MS spectra of a short glycopeptide with a HexNAc. (c) MS/MS spectra of a long glycopeptide with Hex(1)HexNAc(1). (d) MS/MS spectra of a glycopeptide with Hex(2)HexNAc(2).