Probing the O-Glycoproteome of Gastric Cancer Cell Lines for Biomarker Discovery*

Campos, Diana; Freitas, Daniela; Gomes, Joana; Magalhães, Ana; Steentoft, Catharina; Gomes, Catarina; Vester-Christensen, Malene Bech; Ferreira, José Alexandre; Afonso, Luís Pedro; Santos, Lúcio Lara; Pinto-de-Sousa, João; Mandel, Ulla; Clausen, Henrik; Vakhrushev, Sergey Y.; Reis, Celso A.

doi:10.1074/mcp.m114.046862

Cited by 93 publications

(105 citation statements)

References 70 publications

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“…With this approach, we were able to substantially increase our knowledge of the native human O-glycoproteome by unambiguously identifying 1123 sites on 649 proteins, including identification of 541 sites and 231 O-glycoproteins that had not previously been found in native samples. 30,57,60 It should be noted, however, that these data likely underestimate the total number of O-glycans present because of the inherent limitations of shotgun proteomics. 27 This is of particular concern in regards to mucin-like repeat regions and highly homologous protein families that are refractory to mapping by mass spectrometry.…”

Section: Discussionmentioning

confidence: 99%

“…These subpatterns could suggest discrete roles for O-glycans worthy of further investigation; however, such manual analysis is biased by the types of proteins studied, prior knowledge, and potential effects of nonrandom Ser/Thr distribution. Therefore, we performed a systematic and unbiased association study using the full complement of protein annotations available from UniprotKB 54 and all O-glycosites described to date 1,8,[28][29][30]32,47,[55][56][57] (supplemental Table 1). This analysis identifies whether O-glycosites are over-or underenriched around (6 15 amino acids) each protein annotation relative to the expected frequency based on the background distribution of Ser/Thr residues, and is somewhat analogous to a gene ontology study, in which gene ontologies are replaced with protein annotations (Figure 6A).…”

Section: An Unbiased Screen To Identify Protein Features Colocalizingmentioning

confidence: 99%

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Characterizing the O-glycosylation landscape of human plasma, platelets, and endothelial cells

et al. 2017

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Key Points• Human platelets, endothelial cells, and plasma proteins are extensively O-glycosylated, with .1123 O-glycosites identified in this study.• O-glycosites can be classified into functional subgroups; one important function includes the protection from proteolytic processing.The hemostatic system comprises platelet aggregation, coagulation, and fibrinolysis, and is critical to the maintenance of vascular integrity. Multiple studies indicate that glycans play important roles in the hemostatic system; however, most investigations have focused on Using an unbiased screen to identify associations between O-glycosites and protein annotations we found that O-glycans were over-represented close (6 15 amino acids) to tandem repeat regions, protease cleavage sites, within propeptides, and located on a select group of protein domains. The importance of O-glycosites in proximity to proteolytic cleavage sites was further supported by in vitro peptide assays demonstrating that proteolysis of key hemostatic proteins can be inhibited by the presence of O-glycans.Collectively, these data illustrate the global properties of native O-glycosylation and provide the requisite roadmap for future biomarker and structure-function studies.

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Section: Discussionmentioning

confidence: 99%

Section: An Unbiased Screen To Identify Protein Features Colocalizingmentioning

confidence: 99%

Characterizing the O-glycosylation landscape of human plasma, platelets, and endothelial cells

et al. 2017

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“…Finally, the specific interactions of glycans with lectins can also be exploited. Vicia villosa agglutinin has been used to isolate O-glycopeptides from bioengineered cell lines expressing exclusively Tn or sialo Tn antigens [18–22]. Wheat germ agglutinin has been applied for simultaneous isolation of N- and O-glycopeptides from the mouse synaptosome and liver [23, 24].…”

Section: Introductionmentioning

confidence: 99%

O-glycosylation sites identified from mucin core-1 type glycopeptides from human serum

2016

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In this work O-linked glycopeptides bearing mucin core-1 type structures were enriched from human serum. Since about 70% of the O-glycans in human serum bind to the plant lectin Jacalin, we tested a previously successful protocol that combined Jacalin affinity enrichment on the protein- and peptide-level with ERLIC chromatography as a further enrichment step in between, to eliminate the high background of unmodified peptides. In parallel, we developed a simpler and significantly faster new workflow that used two lectins sequentially: wheat germ agglutinin and then Jacalin. The first lectin provides general glycopeptide enrichment, while the second specifically enriches O-linked glycopeptides with Galβ1–3GalNAcα structures. Mass spectrometric analysis of enriched samples showed that the new sample preparation method is more selective and sensitive than the former. Altogether, 52 unique glycosylation sites in 20 proteins were identified in this study.

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“…The O-glycoproteins found in the culture media of these engineered cell models also open for an illustration of which O-glycoproteins are secreted/shed and that have potential to be used as a biomarker. A similar lectin enrichmentbased O-glycoproteomic strategy has therefore also been applied to cancer patient serum samples [39]. In fact, although the cell line models allow for unlimited sample availability and extensive O-glycoproteome analyses, from a biomarker perspective, the use of data from these model systems needs to be combined with data obtained from in vivo settings, such as cancer patients serum and tumor tissues [36].…”

Section: Engineered Cell Models For Characterization Of O-glycoproteomentioning

confidence: 99%

Glycoengineered cell models for the characterization of cancer O-glycoproteome: an innovative strategy for biomarker discovery

Campos

Freitas

Gomes

et al. 2015

Expert Review of Proteomics

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Glycosylation is one of the most abundant forms of protein posttranslational modification. O-glycosylation is a major type of protein glycosylation, comprising different types and structures expressed in several physiologic and pathologic conditions. The understanding of protein attachment site and glycan structure is of the utmost importance for the clarification of the role glycosylation plays in normal cells and in pathological conditions. Neoplastic transformation frequently shows the expression of immature truncated O-glycans. These aberrantly expressed O-glycans have been shown to induce oncogenic properties and can be detected in premalignant lesions, meaning that they are an important source of biomarkers. This article addresses the recent application of genetically engineered cancer cell models to produce simplified homogenous O-glycans allowing the characterization of cancer cells O-glycoproteomes, using advanced mass spectrometry methods and the identification of potential cancer-specific O-glycosylation sites. This article will also discuss possible applications of these biomarkers in the cancer field.

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Probing the O-Glycoproteome of Gastric Cancer Cell Lines for Biomarker Discovery*

Cited by 93 publications

References 70 publications

Characterizing the O-glycosylation landscape of human plasma, platelets, and endothelial cells

Characterizing the O-glycosylation landscape of human plasma, platelets, and endothelial cells

O-glycosylation sites identified from mucin core-1 type glycopeptides from human serum

Glycoengineered cell models for the characterization of cancer O-glycoproteome: an innovative strategy for biomarker discovery

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