Project PXD009476

PRIDE Assigned Tags:
Technical Dataset



Site-specific Extraction of O-linked glycopeptides (EXoO) Maps Landscape of In Vivo O-linked Glycoproteome


Protein glycosylation is one of the most common protein modifications and plays essential roles in biology and therapeutics. However, the analysis of in vivo O-linked glycosylation, a major type of protein glycosylation, has been severely impeded by the scarcity of technology. Here, a chemoenzymatic method was presented for the site-specific extraction of O-linked glycopeptides (EXoO), which achieved simultaneous enrichment and unambiguous mapping of over 3,000 O-linked glycosylation sites and corresponding O-linked glycans on over 1,000 proteins in human kidney tissues, serum and T cells. The large-scale localization of O-linked glycosylation sites nearly doubles the sites identified in the last decades demonstrating that EXoO is the most effective method to-date for defining the site-specific O-linked glycoproteome in different types of sample. Structural analysis of the sites revealed conserved motifs and topological orientation facing extracellular space or lumen of ER and Golgi. Striking signature of aberrant in vivo O-linked glycoproteome was observed between kidney tumor and normal tissues discovering key factors in tumor biology. The O-linked glycoproteome play diverse roles on the ER, Golgi membrane, cell surface and extracellular space arguing that EXoO can be applied broadly to the analysis of O-linked glycoproteins in biology and therapeutics.

Sample Processing Protocol

Solid-phase extraction of site-specific O-linked glycopeptides from fetuin Bovine fetuin (P12763) were denatured in 8 M urea/1 M ammonium bicarbonate (AB) buffer and reduced in 5 mM DTT at 37°C for 1 hour. Proteins were alkylated in 10 mM iodoacetamide at room temperature (RT) for 40 min in the dark. The resulting samples were diluted eight-fold using 100 mM AB buffer before adding trypsin (enzyme/protein ratio of 1/40 w/w) and incubating at 37C for 16 hours. Following digestion, peptides were desalted using a C18 column according to manufacturer’s instructions. Peptides were conjugated to AminoLink resin (Pierce, Rockford, IL) as previously described20. Briefly, the pH of the peptide containing eluate of the C18 column was adjusted to 7.4 by adding phosphate buffer. Peptides were then incubated with the resin (100 µg/100 µl resin, 50% slurry) and 50 mM sodium cyanoborohydride (NaCNBH3) at RT for either at least 4 hours or overnight with shaking. The resin was then blocked by adding 1M Tris-HCl solution (pH7.4) with 50 mM NaCNBH3 at RT for 30 min. The resin was washed three times with 50% acetonitrile, 1.5 M NaCl, and 20 mM Tris-HCl (pH 6.8). The site-specific O-linked glycopeptides were released from the resin by incubation with OpeRATORTM and SialEXOTM (1 unit/1 µg peptides each enzyme, Genovis Inc, Cambridge, MA) in 100 µl of 20 mM Tris-HCl (pH 6.8) at 37C for 16 hours according to manufacturer’s instructions. Following this 16-hour incubation, the released peptides in solution were collected, and the resin was washed twice with 20 mM Tris-HCl to collect the remaining peptides. The pooled peptides were then desalted on a C18 column and dried using lyophilization. Extraction of site-specific O-linked glycopeptides from human kidney tissue, serum and T cells Proteins from human kidney tissues, serum (Sigma-Aldrich, St. Louis, MO) and CEM T cells were trypsin-digested as described above. Following digestion, guanidination of peptides was conducted on a C18 column using procedure described previously to recover the Lys-containing peptides from complex samples while Fetuin does not have Lys in its O-linked glycosylation site containing tryptic peptides17. Briefly, peptides were loaded on a preconditioned C18 column. The column was then sequentially washed three times with 0.1% TFA and guanidination solution (mix equal volume of 2.85 M aqueous ammonia, 0.6 M O-methylisourea and 0.1% TFA, final pH 10.5). After the final wash, guanidination solution was added to cover the C18 material in the column and it was incubated at 65°C for 20 min. Following this incubation, the column was cooled down to RT and washed three time using 0.1% TFA. Peptides were eluted in 60% acetonitrile/0.1% TFA. Intact glycopeptides were enriched using a SAX HyperSepTM Retain AX Columns (RAX) column9. Briefly, after C18 desalting peptides in 60% acetonitrile/0.1% TFA were adjusted to 95% acetonitrile/1% TFA. The RAX column was conditioned in acetonitrile, 100 mM triethylammonium acetate, water and finally 95% acetonitrile/1% TFA for three times per solution. Samples were loaded, washed three times using 95% acetonitrile/1% TFA and eluted in 50% acetonitrile/0.1% TFA. The samples were dried using lyophilization. Peptide fractionation Peptides (100 µg) were fractionated into 96 fractions using a 1220 Series HPLC (Agilent Technologies, Inc., CA) equipped with a Zorbax Extend-C18 analytical column containing 1.8 μm particles at a flow rate of 0.3 ml/min. The mobile-phase A was 10 mM ammonium formate (pH 10) and B was 10 mM ammonium formate and 90% acetonitrile (pH10). Peptides were separated using the following linear gradient: 0–2% B, 10 min; 2–8% B, 5 min; 8–35% B, 85 min; 35–95% B, 5 min; 95–95% B, 15 min. Fractions were collected from 0 to 96 min. The 96 fractions were concatenated into 24 fractions. The samples were dried using lyophilization.

Data Processing Protocol

Database search of site-specific O-linked glycopeptides Bovine fetuin (P12763) in a database with HIV gp120 (AAB50262.1) and TGFbeta1 (P01137) were used for the analysis of fetuin using the same procedure for search of the human protein database except that all fetuin peptides and four glycans including Hex(1)HexNAc(1), HexNAc, Hex(1)HexNAc(2) and Hex(2)HexNAc(2) were used. The RefSeq human protein database (72,956 sequences, downloaded from NCBI website Mar 25, 2015) was used to generate a randomized decoy database (decoy at protein level: decoy-pro) using The Trans-Proteomic Pipeline (TPP). The target and decoy protein database were concatenated and digested on the C-terminal side of Lys/Arg with 2 miss-cleavage sites (trypsin digestion) followed by digested on the N-terminal side of Ser/Thr with 5 miss-cleavage sites (OpeRATOR digestion) in silico and finally Ser/Thr containing peptides with peptide lengths between 6 to 46 amino acids were used resulting in 30,759,520 non-redundant peptide entries. SEQUEST in Proteome Discoverer 2.2 (Thermo Fisher Scientific) was used to search against the database with oxidation (M), guanidination (K), Hex(1)HexNAc(1) (S/T) and HexNAc (S/T) as the variable modifications. Static modification was carbamidomethylation (C). FDR set at 1% using Percolator. MS/MS scan numbers of oxonium ion containing spectra were extracted with 10 ppm tolerance from raw files. Oxonium ion 204 was mandatory together with two of the other oxonium ions. The result was filtered to report identification with glycan modification and oxonium ions in the MS/MS spectra. Percolator generated FDR was inspected using identification labelled with decoy-pro in the output result.


Weiming Yang, Johns Hopkins University
Hui Zhang, Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. ( lab head )

Submission Date


Publication Date


Cell Type

T lymphocyte


Not available


complex glycosylation


Spectrum counting

Experiment Type

Shotgun proteomics


    Yang W, Ao M, Hu Y, Li QK, Zhang H. Mapping the O-glycoproteome using site-specific extraction of O-linked glycopeptides (EXoO). Mol Syst Biol. 2018 14(11):e8486 PubMed: 30459171