Challenges of Determining O-Glycopeptide Heterogeneity: A Fungal Glucanase Model System

Christiansen, M.; Kolarich, Daniel; Nevalainen, Helena; Packer, Nicolle H.; Jensen, Pia

doi:10.1021/ac901717n

Cited by 44 publications

(50 citation statements)

References 40 publications

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“…In general, characterization of complex glycosylation patterns remains extremely challenging (47). CID analysis primarily provides oligosaccharide composition information, with some degree of information on glycan linkages.…”

Section: Fig 4 Etd Spectrum Of 3100 Ngat(galglcnacfuc)c(carbamidomementioning

confidence: 99%

N- and O-Glycosylation in the Murine Synaptosome

Trinidad

Schoepfer²,

Burlingame

et al. 2013

Molecular & Cellular Proteomics

159

230

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We present the first large scale study characterizing both N-and O-linked glycosylation in a site-specific manner on hundreds of proteins. We demonstrate that a lectin-affinity fractionation step using wheat germ agglutinin enriches not only peptides carrying intracellular O-GlcNAc, but also those bearing ER/Golgi-derived N-and O-linked carbohydrate structures. Liquid chromatography-MS (LC/ MS) analysis with high accuracy precursor mass measurements and high sensitivity ion trap electron-transfer dissociation (ETD) were utilized for structural characterization of glycopeptides. Our results reveal both the identity of the precise sites of glycosylation and information on the oligosaccharide structures possible on these proteins. We report a novel iterative approach that allowed us to interpret the ETD data set directly without making prior assumptions about the nature and distribution of oligosaccharides present in our glycopeptide mixture. Over 2500 unique N-and O-linked glycopeptides were identified on 453 proteins. The extent of microheterogeneity varied extensively, and up to 19 different oligosaccharides were attached at a given site. We describe the presence of the well-known mucin-type structures for O-glycosylation, an EGF-domain-specific fucosylation and a rare O-mannosylation on the transmembrane phosphatase Ptprz1. Finally, we identified three examples of O-glycosylation on tyrosine residues. Molecular & Cellular

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Section: Fig 4 Etd Spectrum Of 3100 Ngat(galglcnacfuc)c(carbamidomementioning

confidence: 99%

N- and O-Glycosylation in the Murine Synaptosome

Trinidad

Schoepfer²,

Burlingame

et al. 2013

Molecular & Cellular Proteomics

159

230

View full text Add to dashboard Cite

show abstract

“…Furthermore, O-glycans still prove to be difficult to remove from the protein by both enzymatic and chemical procedures. 11–13 The most common methodology for characterizing proteins with multiple glycosylation sites is therefore by liquid chromatography–tandem mass spectrometry (LC-MS/MS) of protease-generated glycopeptides. Glycosylated species are then identified from masses that correspond to prior known glycopeptides or by searching fragmentation data (MS/MS spectra) against a database containing known peptide and glycan sequences.…”

Section: Introductionmentioning

confidence: 99%

Data-independent oxonium ion profiling of multi-glycosylated biotherapeutics

Madsen

Farutin

Lin

et al. 2018

mAbs

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The characterization of glycosylation is required for many protein therapeutics. The emergence of antibody and antibody-like molecules with multiple glycan attachment sites has rendered glycan analysis increasingly more complicated. Reliance on site-specific glycopeptide analysis is therefore necessary to fully analyze multi-glycosylated biotherapeutics. Established glycopeptide methodologies have generally utilized a priori knowledge of the glycosylation states of the investigated protein(s), database searching of results generated from data-dependent liquid chromatography–tandem mass spectrometry workflows, and extracted ion quantitation of the individual identified species. However, the inherent complexity of glycosylation makes predicting all glycoforms on all glycosylation sites extremely challenging, if not impossible. That is, only the “knowns” are assessed. Here, we describe an agnostic methodology to qualitatively and quantitatively assess both “known” and “unknown” site-specific glycosylation for biotherapeutics that contain multiple glycosylation sites. The workflow uses data-independent, all ion fragmentation to generate glycan oxonium ions, which are then extracted across the entirety of the chromatographic timeline to produce a glycan-specific “fingerprint” of the glycoprotein sample. We utilized both HexNAc and sialic acid oxonium ion profiles to quickly assess the presence of Fab glycosylation in a therapeutic monoclonal antibody, as well as for high-throughput comparisons of multi-glycosylated protein drugs derived from different clones to a reference product. An automated method was created to rapidly assess oxonium profiles between samples, and to provide a quantitative assessment of similarity.

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“…Additionally, the exact glycosylation site of identified peptides containing several Ser/Thr residues cannot be predicted due to the lack of a consensus sequence for mucin-type O-glycosylation. The alternative fragmentation techniques electron capture dissociation (ECD) (38,39) and electron transfer dissociation (ETD) (40) have been introduced for site-specific analysis of CID-labile PTMs but characterization of protein O-glycosylations using ECD/ETD have generally been limited to synthetic glycopeptides or single glycoproteins (41)(42)(43)(44)(45) (46). We have previously developed a sialic acid specific capture-and-release protocol for the enrichment of both N-and O-glycosylated peptides from sialylated glycoproteins in biological samples using hydrazide chemistry (37).…”

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confidence: 99%