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


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.

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Fig. 1: Workflow diagram of EXoO protocol and O-GalNAcylation pathway for biosynthesis of core 1 and 3 O-glycan structures.
Fig. 2: Reproducibility of the EXoO protocol.
Fig. 3: Quantitative performance of the EXoO protocol.
Fig. 4: MS spectra for the determination of OpeRATOR glycan specificity.
Fig. 5: MS2 spectra of O-GalNAc glycopeptides generated using EXoO.

Data availability

The data are available in the PRIDE partner repository48 under the project identifier PXD009476.

Code availability

Extract_oxonium_ion_spectra_intensity.exe script is freely available at GitHub (download at


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This work was supported by the National Institutes of Health, the National Cancer Institute, the Early Detection Research Network (EDRN, U01CA152813), the Clinical Proteomic Tumor Analysis Consortium (CPTAC, U24CA210985) and amfAR, the Foundation for AIDS Research on Bringing Bioengineers to Cure HIV (Grant amfAR 109551‐61‐RGRL).

Author information




W.Y. and H.Z. conceived the research. W.Y., A.S. and Y.X. conducted the experiments and validated the protocol. W.Y., Y.X. and M.A. conducted data analysis.

Corresponding author

Correspondence to Weiming Yang.

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The authors declare no competing interests.

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Key reference using this protocol

Yang, W. et al. Mol. Syst. Biol. 14, e8486 (2018):

Supplementary information

Reporting Summary

Supplementary Data 1

Reproducibility of the EXoO protocol. O-GalNAc glycopeptides were isolated four times from the same 50 µl of human serum. O-GalNAc glycopeptides from one of the isolations were subjected to the same LC–MS/MS analysis for four times to determine the reproducibility of LC–MS/MS analysis. As a result, a total of eight LC–MS/MS runs were conducted, including four isolation repeats and four LC–MS/MS repeats of one of the isolations.

Supplementary Data 2

Quantitative performance of the EXoO protocol.

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Yang, W., Song, A., Ao, M. et al. Large-scale site-specific mapping of the O-GalNAc glycoproteome. Nat Protoc 15, 2589–2610 (2020).

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