Analysis of Mammalian O-Glycopeptides—We Have Made a Good Start, but There is a Long Way to Go

Darula, Zsuzsanna; Medzihradszky, Katalin F.

doi:10.1074/mcp.mr117.000126

Cited by 87 publications

(105 citation statements)

References 146 publications

(189 reference statements)

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“…We observed a marked improvement in glycopeptide and glycoprotein identifications within focused searches, especially for glycopeptides modified with multiple glycans. As a number of unique considerations which are not implemented in open searches, such as accounting for oxonium ions and glycan fragments [13, 73], are needed for optimal glycopeptide identification, this improvement in performance is unsurprising. Consistent with this we observe an increase in the mean Byonic score within focused searches compared to open searches for most datasets (Supplementary figure 5, 8 and 9).…”

Section: Discussionmentioning

confidence: 99%

Open database searching enables the identification and comparison of bacterial glycoproteomes without defining glycan compositions prior to searching

Izaham

Scott

2020

Preprint

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Mass spectrometry has become an indispensable tool for the characterisation of glycosylation across biological systems. Our ability to generate rich fragmentation of glycopeptides has dramatically improved over the last decade yet our informatic approaches still lag behind. While glycoproteomic informatics approaches using glycan databases have attracted considerable attention, database independent approaches have not. This has significantly limited high throughput studies of unusual or atypical glycosylation events such as those observed in bacteria. As such, computational approaches to examine bacterial glycosylation and identify chemically diverse glycans are desperately needed. Here we describe the use of wide-tolerance (up to 2000 Da) open searching as a means to rapidly examine bacterial glycoproteomes. We benchmarked this approach using N-linked glycopeptides of Campylobacter fetus subsp. fetus as well as O-linked glycopeptides of Acinetobacter baumannii and Burkholderia cenocepacia revealing glycopeptides modified with a range of glycans can be readily identified without defining the glycan masses prior to database searching. Utilising this approach, we demonstrate how wide tolerance searching can be used to compare glycan utilisation across bacterial species by examining the glycoproteomes of eight Burkholderia species (B. pseudomallei; B. multivorans; B. dolosa; B. humptydooensis; B. ubonensis, B. anthina; B. diffusa; B. pseudomultivorans). Finally, we demonstrate how open searching enables the identification of low frequency glycoforms based on shared modified peptides sequences. Combined, these results show that open searching is a robust computational approach for the determination of glycan diversity within bacterial proteomes.

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Section: Discussionmentioning

confidence: 99%

Open database searching enables the identification and comparison of bacterial glycoproteomes without defining glycan compositions prior to searching

Izaham

Scott

2020

Preprint

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“…Protein glycosylation is arguably the most diverse and sophisticated form of protein modification which drastically escalates protein heterogeneity to facilitate functional plasticity (Varki, ; Tran & Ten Hagen, ). Compared to N‐linked glycosylation, the study of O‐linked glycosylation has proved difficult due to the structural complexity of the glycans added and the technical challenges posed to definitively characterizing them (Jensen et al , ; Qin et al , ; Darula & Medzihradszky, ). The process of O‐linked glycosylation attaches different O‐linked glycans to Ser and Thr residues, or less commonly Tyr residues in the human proteome (Halim et al , ; Trinidad et al , ).…”

Section: Introductionmentioning

confidence: 99%

“…The process of O‐linked glycosylation attaches different O‐linked glycans to Ser and Thr residues, or less commonly Tyr residues in the human proteome (Halim et al , ; Trinidad et al , ). Among the different types of O‐linked glycosylation seen, O‐linked N‐acetyl‐galactosamine (O‐GalNAc) addition is a major type (Jensen et al , ; Chia et al , ; Darula & Medzihradszky, ). Definitive characterization of O‐linked glycoproteins requires quantitative analysis of O‐linked glycosylation sites and their corresponding glycans.…”

Section: Introductionmentioning

confidence: 99%

“…Definitive characterization of O‐linked glycoproteins requires quantitative analysis of O‐linked glycosylation sites and their corresponding glycans. In contrast to N‐linked glycosylation, where a consensus glycosylation motif has been identified, there is no consensus O‐linked glycosylation motif for the amino acid residues surrounding the glycosylated Ser or Thr (Nishikawa et al , ; Darula & Medzihradszky, ). The cellular machinery for O‐linked glycosylation, located primarily in the Golgi apparatus, is believed to operate stochastically in response to changes in a wide range of both intrinsic and extrinsic factors (Chia et al , ; Bard & Chia, ).…”

Section: Introductionmentioning

confidence: 99%

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Mapping the O‐glycoproteome using site‐specific extraction of O‐linked glycopeptides (EXoO)

Yang

et al. 2018

Molecular Systems Biology

124

181

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Protein glycosylation is one of the most abundant post‐translational modifications. However, detailed analysis of O‐linked glycosylation, a major type of protein glycosylation, has been severely impeded by the scarcity of suitable methodologies. Here, a chemoenzymatic method is introduced for the site‐specific extraction of O‐linked glycopeptides (EXoO), which enabled the mapping of over 3,000 O‐linked glycosylation sites and definition of their glycans on over 1,000 proteins in human kidney tissues, T cells, and serum. This large‐scale localization of O‐linked glycosylation sites demonstrated that EXoO is an effective method for defining the site‐specific O‐linked glycoproteome in different types of sample. Detailed structural analysis of the sites identified revealed conserved motifs and topological orientations facing extracellular space, the cell surface, the lumen of the Golgi, and the endoplasmic reticulum (ER). EXoO was also able to reveal significant differences in the O‐linked glycoproteome of tumor and normal kidney tissues pointing to its broader use in clinical diagnostics and therapeutics.

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“…The workflow describes processing steps in the O-Pair Search strategy, which generates a fragment ion index [1,2] and O-glycan groups [3,4] from user defined protein and O-glycan databases, respectively. Using an ultrafast, fragment-index-enabled open modification search [5] paired with a match of delta masses to aggregate glycan mass combinations [6] enables identification of O-glycopeptide candidates from HCD spectra [7]. Paired EThcD spectra are then used for graph theory-based localization calculations to rapidly assign modification sites for all glycans comprising the O-glycan group [8].…”

Section: Figure 1 O-pair Search Through Metamorpheus For Fast and Comentioning

confidence: 99%

O-Pair Search with MetaMorpheus for O-glycopeptide Characterization

Lü

Riley

Shortreed

et al. 2020

Preprint

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We report O-Pair Search, a new approach to identify O-glycopeptides and localize O-glycosites. Using paired collision-and electron-based dissociation spectra, O-Pair Search identifies Oglycopeptides using an ion-indexed open modification search and localizes O-glycosites using graph theory and probability-based localization. O-Pair Search reduces search times more than 2,000-fold compared to current O-glycopeptide processing software, while defining O-glycosite localization confidence levels and generating more O-glycopeptide identifications. O-Pair Search is freely available: https://github.com/smith-chem-wisc/MetaMorpheus. Main TextMass spectrometry (MS) is the gold standard for interrogating the glycoproteome, enabling the localization of glycans to specific glycosites. 1-3 Recent applications of electron-driven dissociation methods have shown promise in localizing modified O-glycosites even in multiply glycosylated peptides 4 . Yet, standard approaches for interpreting tandem MS spectra are ill-suited for the heterogeneity of O-glycopeptides. Perhaps the most challenging problem for Oglycopeptide analysis is mucin-type O-glycosylation, which is abundant on many extracellular and secreted proteins and is a crucial mediator of immune function, microbiome interaction, and biophysical forces imposed on cells, among others 5 . Mucin-type O-glycans are linked to serine and threonine residues through an initiating N-acetylgalactosamine (GalNAc) sugar, which can be further elaborated into four major core structures (cores 1-4) or remain truncated as terminal GalNAc (Tn) and sialyl-Tn antigens 6 . These O-glycosites occur most frequently in long serine/threonine rich sequences (Supplementary Fig. 1), such as PTS mucin tandem repeat domains, which exist with microheterogeneity defined by a large number of potential O-glycans 7 . The number of serine and threonine residues present in glycopeptides derived from mucin-type O-glycoproteins, combined with the consideration of dozens of potential O-glycans at each site, leads to a combinatorial explosion when generating databases of theoretical O-glycopeptides to consider for each tandem MS/MS spectrum (Supplementary Note 1). AUTHOR INFORMATION Affiliations

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Analysis of Mammalian O-Glycopeptides—We Have Made a Good Start, but There is a Long Way to Go

Cited by 87 publications

References 146 publications

Open database searching enables the identification and comparison of bacterial glycoproteomes without defining glycan compositions prior to searching

Open database searching enables the identification and comparison of bacterial glycoproteomes without defining glycan compositions prior to searching

Mapping the O‐glycoproteome using site‐specific extraction of O‐linked glycopeptides (EXoO)

O-Pair Search with MetaMorpheus for O-glycopeptide Characterization

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