Human urinary glycoproteomics; attachment site specific analysis of N- and O-linked glycosylations by CID and ECD.

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Halim A, Nilsson J, Ruetschi U, Hesse C, Larson G

Human urinary glycoproteomics; attachment site specific analysis of N- and O-linked glycosylations by CID and ECD.

Mol Cell Proteomics. 2012 Apr;11(4):M111.013649. doi: 10.1074/mcp.M111.013649. Epub 2011 Dec 14.

PubMed ID
22171320 [ View in PubMed
]
Abstract

Urine is a complex mixture of proteins and waste products and a challenging biological fluid for biomarker discovery. Previous proteomic studies have identified more than 2800 urinary proteins but analyses aimed at unraveling glycan structures and glycosylation sites of urinary glycoproteins are lacking. Glycoproteomic characterization remains difficult because of the complexity of glycan structures found mainly on asparagine (N-linked) or serine/threonine (O-linked) residues. We have developed a glycoproteomic approach that combines efficient purification of urinary glycoproteins/glycopeptides with complementary MS-fragmentation techniques for glycopeptide analysis. Starting from clinical sample size, we eliminated interfering urinary compounds by dialysis and concentrated the purified urinary proteins by lyophilization. Sialylated urinary glycoproteins were conjugated to a solid support by hydrazide chemistry and trypsin digested. Desialylated glycopeptides, released through mild acid hydrolysis, were characterized by tandem MS experiments utilizing collision induced dissociation (CID) and electron capture dissociation fragmentation techniques. In CID-MS(2), Hex(5)HexNAc(4)-N-Asn and HexHexNAc-O-Ser/Thr were typically observed, in agreement with known N-linked biantennary complex-type and O-linked core 1-like structures, respectively. Additional glycoforms for specific N- and O-linked glycopeptides were also identified, e.g. tetra-antennary N-glycans and fucosylated core 2-like O-glycans. Subsequent CID-MS(3), of selected fragment-ions from the CID-MS(2) analysis, generated peptide specific b- and y-ions that were used for peptide identification. In total, 58 N- and 63 O-linked glycopeptides from 53 glycoproteins were characterized with respect to glycan- and peptide sequences. The combination of CID and electron capture dissociation techniques allowed for the exact identification of Ser/Thr attachment site(s) for 40 of 57 putative O-glycosylation sites. We defined 29 O-glycosylation sites which have, to our knowledge, not been previously reported. This is the first study of human urinary glycoproteins where "intact" glycopeptides were studied, i.e. the presence of glycans and their attachment sites were proven without doubt.

DrugBank Data that Cites this Article

Polypeptides
NameUniProt ID
ProthrombinP00734Details
Coagulation factor XP00742Details
Vitamin K-dependent protein CP04070Details
Alpha-1-acid glycoprotein 1P02763Details
Protein AMBPP02760Details
Alpha-1-antitrypsinP01009Details
Pro-epidermal growth factorP01133Details
Basement membrane-specific heparan sulfate proteoglycan core proteinP98160Details
CD44 antigenP16070Details
Plasma serine protease inhibitorP05154Details
Syndecan-2P34741Details
Plasma protease C1 inhibitorP05155Details
Zinc-alpha-2-glycoproteinP25311Details
Carboxypeptidase B2Q96IY4Details
Alpha-1-acid glycoprotein 2P19652Details
Poliovirus receptorP15151Details
Apolipoprotein DP05090Details
Ig alpha-1 chain C regionP01876Details
Alpha-2-HS-glycoproteinP02765Details
Inter-alpha-trypsin inhibitor heavy chain H2P19823Details
Inter-alpha-trypsin inhibitor heavy chain H4Q14624Details
Prostaglandin-H2 D-isomeraseP41222Details
CD27 antigenP26842Details