Dataset Information

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

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.

SUBMITTER: Halim A

PROVIDER: S-EPMC3322569 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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

Halim Adnan A Nilsson Jonas J Rüetschi Ulla U Hesse Camilla C Larson Göran G

Molecular & cellular proteomics : MCP 20111214 4

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 ...[more]

PMID: 22171320

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Project description:Epithelial cells lining the urinary tract secrete urinary exosomes (40-100 nm) that can be targeted to specific cells modulating their functionality. One potential targeting mechanism is adhesion between vesicle surface glycoproteins and target cells. This makes the glycopeptide analysis of exosomes important. Exosomes reflect the physiological state of the parent cells; therefore, they are a good source of biomarkers for urological and other diseases. Moreover, the urine collection is easy and noninvasive and urinary exosomes give information about renal and systemic organ systems. Accordingly, multiple studies on proteomic characterization of urinary exosomes in health and disease have been published. However, no systematic analysis of their glycoproteomic profile has been carried out to date, whereas a conserved glycan signature has been found for exosomes from urine and other sources including T cell lines and human milk. Here, we have enriched and identified the N-glycopeptides from these vesicles. These enriched N-glycopeptides were solved for their peptide sequence, glycan composition, structure, and glycosylation site using collision-induced dissociation MS/MS (CID-tandem MS) data interpreted by a publicly available software GlycopeptideId. Released glycans from the same sample was also analyzed with MALDI-MS. We have identified the N-glycoproteome of urinary exosomes. In total 126 N-glycopeptides from 51 N-glycosylation sites belonging to 37 glycoproteins were found in our results. The peptide sequences of these N-glycopeptides were identified unambiguously and their glycan composition (for 125 N-glycopeptides) and structures (for 87 N-glycopeptides) were proposed. A corresponding glycomic analysis with released N-glycans was also performed. We identified 66 unique nonmodified N-glycan compositions and in addition 13 sulfated/phosphorylated glycans were also found. This is the first systematic analysis of N-glycoproteome of urinary exosomes.

Project description:Glycosylation is a common protein modification with a significant role in many vital cellular processes and human diseases, making the characterization of protein-attached glycan structures important for understanding cell biology and disease processes. Direct analysis of protein N-glycosylation by tandem mass spectrometry of glycopeptides promises site-specific elucidation of N-glycan microheterogeneity, something that detached N-glycan and deglycosylated peptide analyses cannot provide. However, successful implementation of direct N-glycopeptide analysis by tandem mass spectrometry remains a challenge. In this work, we consider algorithmic techniques for the analysis of LC-MS/MS data acquired from glycopeptide-enriched fractions of enzymatic digests of purified proteins. We implement a computational strategy that takes advantage of the properties of CID fragmentation spectra of N-glycopeptides, matching the MS/MS spectra to peptide-glycan pairs from protein sequences and glycan structure databases. Significantly, we also propose a novel false discovery rate estimation technique to estimate and manage the number of false identifications. We use a human glycoprotein standard, haptoglobin, digested with trypsin and GluC, enriched for glycopeptides using HILIC chromatography, and analyzed by LC-MS/MS to demonstrate our algorithmic strategy and evaluate its performance. Our software, GlycoPeptideSearch (GPS), assigned glycopeptide identifications to 246 of the spectra at a false discovery rate of 5.58%, identifying 42 distinct haptoglobin peptide-glycan pairs at each of the four haptoglobin N-linked glycosylation sites. We further demonstrate the effectiveness of this approach by analyzing plasma-derived haptoglobin, identifying 136 N-linked glycopeptide spectra at a false discovery rate of 0.4%, representing 15 distinct glycopeptides on at least three of the four N-linked glycosylation sites. The software, GlycoPeptideSearch, is available for download from http://edwardslab.bmcb.georgetown.edu/GPS .

Dataset Information

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

Publications

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

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